the first collaborative meeting on phytomedicine...committees & secretariat program at a glance...

http://www.phyto-medicine.eu/

THE FIRST COLLABORATIVE MEETINGON PHYTOMEDICINE

Development and Clinical Validationof Medicinal Plants for Rational Pharmacotherapy

Alpine Foundation for Life Sciences of Olivone, CHUniversity of Applied Sciences HES-SO Valais/Wallis, CH

University of Geneva, CHUniversity of Heidelberg, DUniversity of Insubria, IUniversity of Zürich, CH

Monte Verità, Ascona - Switzerland11th –13th May 2007

http://www.phyto-medicine.eu/index.php

Final Program&

Abstract Book


THE FIRST COLLABORATIVE MEETING ON PHYTOMEDICINEDevelopment and Clinical Validation of Medicinal Plants for Rational Pharmacotherapy

Monte Verità, Ascona – Switzerland, 11th –13th May 2007

Organized by

Under Auspices of

Comunità di Lavoro Regio Insubrica

Repubblica e Cantone Ticino

Provincia di Varese

Università degli Studi dell'Insubria

Ordine dei Medici Chirurghi e degli Odontoiatri della provincia di Varese

Ordine dei Medici e Odontoiatri di Como

Ordine dei Medici del Cantone Ticino




THE FIRST COLLABORATIVE MEETINGON PHYTOMEDICINE

Development and Clinical Validationof Medicinal Plants for Rational Pharmacotherapy

Alpine Foundation for Life Sciences of Olivone, CHUniversity of Applied Sciences HES-SO Valais/Wallis, CH

University of Geneva, CHUniversity of Heidelberg, DUniversity of Insubria, IUniversity of Zürich, CH

Monte Verità, Ascona - Switzerland11th –13th May 2007

http://www.phyto-medicine.eu/index.php

Final Program&

Abstract Book

Committees & Secretariat

Program at a Glance

Final Program

General Information

ABSTRACTS

Plenary Lectures

Session 1Medicinal plants and cancer

Session 2Medicinal plants and immune related diseases

Session 3Medicinal plants and neurodegenerative diseases

Session 4Medicinal plants and safety

Session 5Methodological issues in clinical trials with medicinal plants

Poster session

Index of Authors




Index

02

03

05

09

12

13

20

27

31

40

43

47

85

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Scientific Committee

Rudolf Bauer, AustriaArio Conti, SwitzerlandMarco Cosentino, ItalyFederico Dajas, Uruguay

Theodor Dingermann, GermanyKurt Hostettmann, Switzerland

Sergio Lecchini, ItalyFranca Marino, Italy

Silvano Paracchini, SwitzerlandJürgen Reichling, GermanyReinhard Saller, SwitzerlandGianni Soldati, Switzerland

Jean-Claude Villettaz, SwitzerlandAngelika Vollmer, GermanyHeinrich Walt, SwitzerlandMichael Wink, Germany

Local Organising Committee

Ario Conti (Co-Chairman), SwitzerlandMarco Cosentino (Co-Chairman), Italy

Raffaella Bombelli, ItalyMarco Ferrari, ItalyFranca Marino, ItalyEmanuela Rasini, Italy

Gianni Soldati, Switzerland

Secretariat

Mrs. Paola GervasiniDepartment of Clinical MedicineSection of Experimental and Clinical PharmacologyUniversity of Insubria, Varese – ItalyTel.: +39 0332 217401Fax: +39 0332 217409E-mail: [email protected]



Mrs. Santina BerettaAlpine Foundation for Life Sciences

Olivone - SwitzerlandTel.: +41 (0)91 872 21 68Fax: +41 (0)91 872 21 69

E-mail: [email protected]

02

Program at a Glance

16.00

16.15 – 17.00

17.00

9.00 – 9.45

9.45 – 10.45

10.45 – 11.00

11.00 – 11.45




Opening Ceremony

Plenary lecture: Nullis amor est medicabilis herbis - From the certainties of ancient science to the problems of contemporary medicine

Welcome Drink

Lecture I: The potential of Alpine plants as source of new drugs

Session 1: Medicinal plants and cancer

Break

Lecture II: Breeding and optimising the cultivation procedures of medicinal plants to improve their benefits

Friday 11th May 2007

Saturday 12th May 2007

11.45 – 12.45

12.45 – 14.15

14.15 – 15.00

15.00 – 16.45

Session 2: Medicinal plants and immune related diseases

Break

Lecture III: Molecular mode of action of drugs used in phytomedicine

Session 3: Medicinal plants and neurodegenerative diseases.

16.45 – 17.00

17.00 – 17.40

17.40 – 18.40

20.00

Break

Session 4: Medicinal plants and safety

Poster session – guided tour

Social Dinner

03

11.00 – 11.45

11.45 – 12.00

12.00




Round Table: Development and clinical validation of medicinal plants for rational pharmacotherapy

Closing Ceremony

Farewell Lunch

9.00 – 9.45

9.45 – 10.45

10.45 – 11.00

Lecture IV: Lead structure discovery using ethnopharmacological approaches and virtual screening techniques

Session 5: Methodological issues in clinical trials with medicinal plants

Break

Sunday 13th May 2007

04

Final Program

16.00

16.15 – 17.00

17.00

9.00 – 9.45

9.45 – 10.45

10.45 – 11.00

11.00 – 11.45




Opening Ceremony

Plenary lecture: Nullis amor est medicabilis herbis - From the certainties of ancient science to the problems of contemporary medicineGiuseppe Armocida (Italy)

Welcome Drink

Lecture I: The potential of Alpine plants as source of new drugsKurt Hostettmann (Switzerland)Chairman – Ario Conti

Session 1: Medicinal plants and cancerChairman – Clarissa Gerhäuser, Heinrich Walt

Selected plant extracts and secondary metabolites inducing apoptosis in human cancer cellsKatarina Hostanska (Switzerland)Secondary plant metabolites from apples and colon cancer preventionClarissa Gerhäuser (Germany)Artemisinin and its derivatives - Novel treatment options for HPV-infection and proliferative cervical disordersAstrid Baege (Switzerland)

Break

Lecture II: Breeding and optimising the cultivation procedures of medicinal plants to improve their benefitsChristoph Carlen (Switzerland)Chairman – Jean-Claude Villettaz

Friday 11th May 2007

Saturday 12th May 2007

05

11.45 – 12.45

12.45 – 14.15

14.15 – 15.00

15.00 – 16.45




Session 2: Medicinal plants and immune related diseasesChairmen – Rudolf Bauer

Recent progress in Echinacea researchRudolf Bauer (Austria)Immunomodulatory alkylamides in Echinacea, what is their role?Jürg Gertsch (Switzerland)Immunomodulatory, pharmacological and therapeutic properties of MenthaMarco Cosentino (Italy)

Break

Lecture III: Molecular mode of action of drugs used in phytomedicineMichael Wink (Germany)Chairman – Jürgen Reichling

Session 3: Medicinal plants and neurodegenerative diseases.Chairmen – Federico Dajas, Pierre-Alain Carrupt

Neuroprotective activity of curcumin in in vitro models of neurodegeneration. Its potential as a therapeutic tool against neurodegenerationFiorella Malchiodi-Albedi (Italy)Mechanisms of action of polyphenols in biological systems: effects on intracellular redox balanceRoberta Masella (Italy)Dissection of the cell signalling pathway implicated in the mechanisms of neuroprotection afforded by the essential oil of bergamot against excitotoxic neuronal deathM. Tiziana Corasaniti, Simona Maida, Vincenza Fratto, Michele Navarra and Giacinto Bagetta (Italy)From medicinal plants to multifunctional therapy for neurodegenerative diseases: pharmacodynamic and pharmacokinetic challengesPierre-Alain Carrupt (Switzerland)The neuroprotective capacity of some Asteraceae and their polyphenol moleculesFederico Dajas (Uruguay)

06

16.45 – 17.00

17.00 – 17.40

17.40 – 18.40

20.00

9.00 – 9.45

9.45 – 10.45

10.45 – 11.00




Break

Session 4: Medicinal plants and safetyChairman – Jürgen Reichling

HET-CAM-assay - a non-animal approach to assess mucous membrane irritation of essential oilsJürgen Reichling (Germany)Molecular mechanism of cancer caused by Aristolochia plantsHeinz Schmeiser (Germany)

Poster session – guided tour

Social Dinner

Lecture IV: Lead structure discovery using ethnopharmacological approaches and virtual screening techniquesHermann Stuppner (Austria)Chairman – Marco Cosentino

Session 5: Methodological issues in clinical trials with medicinal plantsChairmen – Heinrich Walt, Marco Cosentino

Phytomedicine IS scientific medicine: What’s next?Heinrich Walt (Switzerland)From standard Tanacetum Parthenium extract to a stabilized derivative of parthenolide: studies in animal models of painCristina Tassorelli, Paolo Morazzoni (Italy)Methodology and reporting of clinical trials of herbal medicine interventionsJoel Gagnier (Canada)

Break

Sunday 13th May 2007

07

11.00 – 11.45

11.45 – 12.00

12.00




Round Table: Development and clinical validation of medicinal plants for rational pharmacotherapyArio Conti (Switzerland)Marco Cosentino (Italy)Joel Gagnier (Canada)Paolo Morazzoni (Italy)Silvano Paracchini (Switzerland)Cristina Tassorelli (Italy)Heinrich Walt (Switzerlend)

Closing Ceremony

Farewell Lunch

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General Information

REGISTRATION AND PAYMENT INFORMATION

Advance registration is required by 2nd April 2007 to guarantee the availability of conference materials.

We encourage all participants to stay for the whole duration of the meeting. The fixed registration fee will be charged regardless of the length of participation.

Please transfer your registration fee to:

Alpine Foundation for Life Sciences6718 Olivone, Switzerland

IBAN CH92 8035 0000 0022 8891 7; SWIFT code RAIFCH22Name and address of bank:

Banca Raiffeisen6718 Olivone, Switzerland

stating “Phytomedicine Meeting Ascona 2007”

Acknowledgement of registration will be issued via e-mail or fax prior to the conference. The maximum number of participants is 200, so please apply soon.

REGISTRATION FEES

CATEGORY UNTIL 19th Feb UNTIL 2nd April ON SITE Delegate € 150,00 € 180,00 € 240,00Student € 90,00 € 120,00 € 160,00Accompanying € 70,00 € 90,00 € 120,00Person

Registration fee for Participants and Students entitles to attend the lectures and poster session and includes: conference book, welcome drink (11 May), social dinner (12 May), coffe break and lunch (12, 13 May).

Registration fee for Accompanying Persons excludes the lectures and poster session and includes: welcome drink (11 May), social dinner (12 May) and lunch (12, 13 May).

Students must present registration signed and stamped by the Principal, Dean, or Rector of the origin institution of studies.

ACCOMMODATION

The cost of the accommodation is not included in the Registration fee. Please refer to next section for information about Hotel reservation.

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Hotel Reservation

Single/double rooms for Meeting participants and accompanying persons have been blocked in 3/4 star Hotels in the town of Ascona.

Reserving accommodation at special rates will be possible up to 19th March 2007.

For Hotel reservation please contact:

Congressi Monte Verità6612 AsconaSwitzerland

Tel. +41 (0)91 785 40 40Fax +41 (0)91 785 40 [email protected]

Social Program

Friday 11th May 2007 Welcome DrinkSaturday 12th May 2007 Social Dinner

Secretariat

Mrs. Paola GervasiniDepartment of Clinical MedicineSection of Experimental and Clinical PharmacologyUniversity of Insubria, Varese – ItalyTel.: +39 0332 217401Fax: +39 0332 217409E-mail: [email protected]

(registration and scientific program)

Further Information

The final program of the Meeting will be mailed to registered participants by 23rd April 2007. Updated information can be found at the website of the Meeting

www.phyto-medicine.eu/index.php

Deadline Dates

Reduced Registration Fee 19th February 2007Reserving Accommodation at Special Rates 19th March 2007Receipt of Registration Form 2nd April 2007Receipt of Abstracts 2nd April 2007Notification of Abstract Acceptance 16th April 2007

Mrs. Santina BerettaAlpine Foundation for Life Sciences

Olivone - SwitzerlandTel.: +41 (0)91 872 21 68Fax: +41 (0)91 872 21 69

E-mail: [email protected]

(registration fees)



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VenueThe First Collaborative Meeting on Phytomedicine will be hosted by the Monte Verità Centre from friday 11th May to Sunday 13th May 2007.Monte Verità is set amidst the hills overlooking Ascona and Lake Maggiore. From the very beginning it has been a magnet for the convergence of ideas, movements, experiments and historical personages. Further information about the Monte Verità Centre can be found at the Centre website http://www.monteverita.org/

LanguageThe official language of the Meeting is English. No simultaneous translation will be provided.

How to Reach AsconaTown of Ascona http://www.ascona.ch/Lake Maggiore Tourism Office http://www.maggiore.ch/Lugano (CH) Airport http://www.lugano-airport.ch/Milan (I) Airports http://www.sea-aeroportimilano.it/Swiss Railways http://www.sbb.ch/en/



Monte Verità CentreAscona CH

Lugano (CH) AirportAgno

Milan (I) AirportLinate

Milan (I) AirportMalpensa

Locarno (CH)Railway Station

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Abstracts

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Plenary Lectures

Nullis amor est medicabilis herbis - From the certainties of ancient science to the problems of contemporary medicineGiuseppe Armocida (Italy)

Breeding and optimising the cultivation procedures of medicinal plants to improve their benefitsChristoph Carlen (Switzerland)

The potential of Alpine plants as source of new drugsKurt Hostettmann (Switzerland)

Lead structure discovery using ethnopharmacological approaches and virtual screening techniquesHermann Stuppner (Austria)

Molecular mode of action of drugs used in phytomedicineMichael Wink (Germany)



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NULLIS AMOR EST MEDICABILIS HERBIS - FROM THE CERTAINTIES OF ANCIENT SCIENCE TO THE PROBLEMS OF CONTEMPORARY MEDICINE

Giuseppe Armocida

President of the Italian Society of History of Medicine, Head of the Department of Medicine and Public Health, University of Insubria



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BREEDING AND OPTIMISING THE CULTIVATION PROCEDURES OF MEDICAL PLANTS TO IMPROVE THEIR BENEFITS

CARLEN Christoph1, MALNOE Pia1, BAROFFIO Catherine1, SIMONNET Xavier2

1 Agroscope Changins-Wädenswil Research Station ACW; 2 Mediplant

Plants are the source of many important pharmaceuticals. An important issue for human health is to optimise the production of the valuable secondary plant metabolites. Considerable effort has been made to generate such metabolites in plant cell or tissue culture. Nevertheless, horticultural production of medical plants still remains the main supply for plant–derived pharmaceuticals. Agronomic research plays therefore an essential role to optimise production of medicinal plants. The importance of breeding programmes and optimising cultural cultivation procedures to improve the benefit of medical plants is revealed by some projects conduced by Agroscope Changins-Wädenswil Research Station ACW and Mediplant.

Breeding for increased levels of wanted compounds in plants allowed to obtain cultivars of Artemisia annua L. with content of artemisinin, a highly potent antimalarial compound, nearing 2 %. Only cultivars with a high artemisinin production potential permit to answer to the increasing demand for this molecule. Breeding for abiotic and biotic tolerance increases the stability of supply. As an example, Hypericum perforatum L., a plant with anti-depressor compounds, suffers badly by a pathogen called Dieback. A breeding programme resulted in an obtention of an cultivar which showed a good tolerance to this disease and a good productivity in flowering segments, thus enabling an improved cost efficient cultivation. Breeding for better homogeneity of crops, such as for Salvia officinalis L., resulted in the new cultivar Regula, allowing to better standardise the production and to increase the quality of the harvested plants. Breeding for elimination of unwanted compounds is also an important topic. Agroscope ACW created a cultivar of Artemisia umbelliformis Lam. without thujone (neurotoxic molecules).

To optimise the quality of the plant material used for pharmaceuticals, research on best practices of cultivation are also important such as planting, fertilisation, irrigation, harvest period and harvest as well as drying techniques. Experiments on the ideal stage of harvest showed for A. umbelliformis and Tanacetum vulgare L. that the best harvest period for is the beginning of flowering, For S. officinalis,



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trials with different cutting procedures showed that 3 harvests per year with a last cut at the latest towards the beginning of September at a height of 15 cm, enhances the quality, the winterfrost resistance and consequently productivity in mountain areas. Such research results are integrated to the guidelines for GAP (good agricultural practice) recommending cultivation procedure to optimise the quality of medicinal plants.



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THE POTENTIAL OF ALPINE PLANTS AS SOURCE OF NEW DRUGS

Kurt Hostettmann

Laboratory of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne

The Alpine flora is unique as its plants grow at high altitude and are submitted to various stress conditions, such as cold temperatures and intense UV radiations. Since the wellknown edelweiss could be domesticated, the extracts of this species are widely used for their radical scavenging properties. Gentians, other typical Alpine plants, known since the Antiquity to facilitate digestion, present a potentiel to fight depression and Alzheimer’s disease. Recently, golden root or Rhodiola rosea L. (Crassulaceae) became very famous after an article published in 2003 in Newsweek called the plant a « herbal stress buster ». The plant is traditionally used to fight fatigue and to enhance performance. Recent studies indicate that Rhodiola rosea L. is an adaptogen inducing more rapid effects than the wellknown ginseng. Cultivation of this highly promising plant are currently undertaken in Switzerland. Alpine fruits such as Vaccinium myrtillus L. (blueberry) are used to fight diarrhea and to improve blood circulation and night vision and Vaccinium vitis-idaea L. is used more and more, with other related species such as cranberry for the treatment of infection of the urinary tract and cystites. The Alpine flora is still an unexplored reservoir of new molecules to be discovered.



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LEAD STRUCTURE DISCOVERY USING ETHNOPHARMACOLOGICAL APPROACHES AND VIRTUAL SCREENING TECHNIQUES

H. Stuppner1, J.M. Rollinger1, T. Langer2

Institute of Pharmacy/Pharmacognosy1, Institute of Pharmacy/CAMD2 group, Leopold-Franzens-University, Innrain 52c, 6020 Innsbruck, Austria

The potency of natural products (NPs) to treat and cure human diseases is well-known and has been for living memory. Even today, in the century of combinatorial chemistry, secondary metabolites of plants, fungi, marine organisms, and microorganisms are still an important source for the development of new drugs. About 50% (Newman and Cragg, 2007) of the drugs introduced to the market during the last 25 years have been derived directly or indirectly from small molecules of natural origin. However, it remains an exciting challenge to target and efficiently select lead (active) compounds from the multitude and biodiversity of natural products. For the efficient discovery of plant constituents with interesting pharmacological properties we follow up different strategies in our laboratory: i) random screening by means of bioassays; ii) targeted search using leads from ethnopharmacology and iii) computer-aided drug discovery combining pharmacophore modelling with virtual screening. The latter include the prediction of bioactive constituents (i.e. virtual hits) by means of pharmacophore-based virtual screening (VS) and docking. Access is provided for two in-house generated 3D multiconformational databases, which are basic requirements for the VS process: The NP-database consists of more than 110.000 NPs and the database DIOS contains about 10.000 constituents of medicinal plants originating from the ethnopharmacological source ‘de materia medica’ by Pedanius Dioscorides (1st cent. AD).It is evident that the in silico approach can offer only virtual hits which are sometimes rather speculative. Therefore, a sensible combination with established strategies from classical pharmacognosy (in combo approach) is advisable, which may offer a more deepened access to bioactive NPs. Some basic principles, requirements and limitations of virtual screening strategies will be demonstrated and their applicability in NP research confirmed by selected examples. Integrated virtual screening workflows will be outlined.

Newman, D; Cragg, G; Natural Products as Sources of New Drugs over the Last 25 Years. J Nat Prod 2007



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MOLECULAR MODES OF ACTION OF DRUGS USED IN PHYTOMEDICINE

Michael Wink

University of Heidelberg, IPMB, INF 364, 69120 Heidelberg, Germany

Plant medicines usually contain complex mixtures of secondary metabolites, often consisting of polyphenols and terpenoids. Some extracts contain active agents that specifically interact with a cellular receptor. However, most ingredients can be regarded as broadband active compounds with pleiotropic effects that can interact with proteins and biomembranes of human cells by forming non-covalent bonds. In addition, also covalent modifications of proteins can be caused by mustard oils, aldehydes, epoxides, sesquiterpene lactones, allicin or iridoids. These interactions influence the conformation of proteins and thus their activity and possibility for cross talk with other proteins. Since many disorders are due to disturbances in several proteins, the unspecific covalent and non-covalent protein modifications by secondary metabolites is of special importance and seems to be the base how many plant medicines work.



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Session 1

Medicinal plants and cancer

Selected plant extracts and secondary metabolites inducing apoptosis in human cancer cellsKatarina Hostanska (Switzerland)

Secondary plant metabolites from apples and colon cancer preventionClarissa Gerhäuser (Germany)

Artemisinin and its derivatives - Novel treatment options for HPV-infection and proliferative cervical disordersAstrid Baege (Switzerland)



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SELECTED PLANT EXTRACT AND SECONDARY METABOLITES INDUCING APOPTOSIS IN HUMAN CANCER CELLS

Katarina Hostanska

University Hospital Zurich, Department of Internal Medicine, Institute for Complementary Medicine,Raemistrasse 100, 8091 Zurich, Switzerland; e-mail: [email protected]

Plant extracts have been used to alleviate various diseases for thousands of years. Recently, there have been extensive efforts to employ scientific methods to prove the efficacy of many of these herbs. Cancer is a major public health problem. Breast cancer is the most common malignancy affecting women and prostate carcinoma is the most common male malignancy in many industrialized Western countries. Lung and colon cancer are the most common cancer-related cause of death among men and women worldwide. Chemoprevention has emerged as an exceedingly important prevention objective for many forms of cancer. Chemopreventive agents must be safe and well tolerated for chronic usage, and ideally, they should be relatively cost effective. An increasing variety of natural products are investigated for their anticancer activity.

Apoptosis plays a critical role in the multiple steps (transformation, progression and survival of metastases) of tumorigenesis. The modulation of the apoptotic response of human cancer cells provides new hope for therapeutic strategies in these diseases. We turned our attention on hormone-depedent , epidemiologically important tumor diseases, breast and prostate cancer. Hormone replacement therapy is contraindicated in women with breast cancer. Extracts from rhizomes of black cohosh Actaea syn. Cimicifuga racemosa (CR), have gained acceptance as a natural alternative for the treatment and prevention of menopausal disorders. We investigated the antiproliferative and apoptosis inducing effect of an isopropanolic-aqueous extract of black cohosh and its two major fractions the triterpene glycosides (TTG) or cinnamic acid esters (CAE) on estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA MB231breast cancer cells, as well as on androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells. Black cohosh extract kills human hormone-responsive or-unresponsive breast and prostate cancer cells by induction of apoptosis and activation of caspases. TTG and CAE compounds significantly contributed to its apoptotic effect, CAE being the more potent inhibitor of



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proliferation and apoptosis inducer. In addition, the invasive capacity of highly metastatic MDA-MB231 cells was suppressed by 50% at an non cytotoxic extract concentration (1-4).Conclusion: Antiinvasive activity together with the antiproliferative and apoptosis-inducing effect of iCR suggest its use as a secure agent in postmenopausal hormone replacement therapy with additional chemopreventive activity.Nonsteroidal anti-inflammatory drugs (NSAID) have become the focus of research investigating their potential prevention. Willow bark (Salix alba), which has been used throughout the world for centuries as an antipyretic and analgesic, had comparable antiinflammatory activities as acetyl-salicylic acid. Overexpression of cyclooxygenase-2 (COX-2) has been observed in colon and lung tumors.Considering the inhibition of prostaglandin synthesis (PGs) as a key determinant by the chemopreventive and antineoplastic effect of NSAIDs, we investigated representative colon (HT29, HCT116) and lung (A549, SW2) cancer cell lines from each group (COX-2 proficient, COX-2 deficient) for their sensitivity to willow bark extract and its salicylalcohol derivates (F1), flavonoids (F2) and proanthocyanidins (F3) fractions. Willow bark extract and its fractions promote apoptosis in human colon and lung cancer cell lines irrespective their COX-2, p53 and Bcl-2 status (5).Conclusion: Antiproliferative and apoptosis-inducing effects of willow bark extract make it an interesting option in colon and lung cancer warranting further in vitro and in vivo investigations.

References: 1. Hostanska K et al., (2004) Breast Cancer Res Treat 84 : 151-160; 2. Hostanska et al., (2004) Biol Pharm Bull 27: 1970-1975; 3. Hostanska et al., (2005) Anticancer Res 25: 139-148; 4. Hostanska et al., in vivo (2007) 21: in press; 5.Hostanska et al., (2007) Cancer Detect Prev (2007) 31: in press



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SECONDARY PLANT METABOLITES FROM APPLES AND COLON CANCER PREVENTION

Clarissa Gerhauser,1 Lydia Pan,1 Henriette Zessner,2 Frank Will,3 Karin Klimo,1

Norbert Frank,1 Helmut Dietrich,3 Helmut Bartsch,1 Hans Becker2

1German Cancer Research Center, Heidelberg, Germany; 2University of Saarland, Saarbruecken, Germany; 3Research Institute Geisenheim, Geisenheim, Germany.

Carcinomas of the gastrointestinal tract are the second leading cause of cancer-related death in industrialized countries. Epidemiological studies reveal a strong dietary influence on colorectal cancer incidence. It is well established that a diet rich in food and vegetables contributes to a cancer preventive lifestyle. Apples and apple juice are widely consumed and a rich source of phytochemicals with potential cancer preventive potential.

The aim of the present study was to investigate colon cancer preventive efficacy of apple juice in comparison with a polyphenol-enriched apple extract in ApcMin

mice that develop multiple intestinal neoplasia due to a mutation in the Apc(adenomatous polyposis coli) gene. This model has been used to demonstrate colon cancer preventive potential of a variety of dietary phytochemicals, including curcumin from turmeric, resveratrol from grapes, catechin and epigallocatechin gallate from green tea, caffeic acid phenethylester (CAPE) from propolis, anthocyanins from cherries, sulforaphane from broccoli (http://corpet.net/min).

Dietary intervention with apple juice or extract (0.2% in drinking water) for 10 weeks significantly lowered the number of adenomas in the small intestine by 38% and 40%, respectively, whereas tumor numbers in the colon were not affected. Clinical parameters, i.e. reduced hematocrit values as an indication of intestinal bleeding, and increased spleen weights were ameliorated by apple juice and especially extract treatment.

Apple juice is known to contain several classes of phenolic compounds, including phenol carbonic acids, dehydrochalcones, flavonoids, catechins and procyanidins. To further characterize apple juice components which might account for the observed cancer preventive effects, apple juice extracts obtained by solid phase extraction were tested in a series of in vitro bioassays relevant for inhibition of carcinogenesis in vivo, and subjected to activity-guided fractionation. In vitro screening of extract and fractions revealed a broad spectrum of colon cancer



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preventive properties, including antioxidant and radical scavenging activity, potential to modulate xenobiotic metabolism, as well as anti-inflammatory, anti-hormonal and anti-proliferative effects. Comparison of these results with those obtained with more than 20 isolated apple constituents indicated that distinct classes of polyphenols account for specific activities, but these did not fully explain the preventive potential obtained with complex apple extracts. We hypothesize that the combination of chemopreventive mechanisms mediated by apple juice components contributes additively or synergistically to the cancer inhibitory potential. Overall, oligomeric procyanidins were identified as the most potent class of constituents.

Apples and apple juice are an integral part of the human diet and are consumed by a majority of the population, including children. Our study and others (1,2)

suggest that apple juice and apple extracts should be further investigated as part of a prevention strategy for hereditary and sporadic colorectal cancer.

References:1. Gosse, F. et al. Chemopreventive properties of apple procyanidins on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis. Carcinogenesis 26, 1291-1295 (2005).2. Barth, S.W. et al. Cloudy apple juice decreases DNA-damage, hyperproliferation and aberrant crypt foci development in the distal colon of DMH-initiated rats. Carcinogenesis 26, 1414-1421 (2005).



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ARTEMISININ AND ITS DERIVATIVES – NOVEL TREATMENT OPTIONS FOR HPV-INFECTION AND PROLIFERATIVE CERVICAL DISORDERS

Astrid C Baege1, Richard Schlegel2, Daniel Fink1 and Gary H Posner3

1 Dept. of Gynecology, Medical Center of the University of Zurich, Zurich, Switzerland2 Dept. of Pathology, Georgetown University Medical Center, Washington DC, USA3 Dept. of Chemistry, Johns Hopkins University, Baltimore, MD, USA

Cervical cancer is still the 2nd most common malignancy related cause of death in women worldwide. Although population wide screening in most Western countries has led to a remarkable reduction in incidence and mortality, with approximately 470,000 new cases diagnosed each year cervical cancer remains a global public health problem and a significant economic burden to health care systems. Nearly all cervical cancers are etiologically attributable to persistent high risk human papillomaviruses (HPV) infection. Potent antiviral agents to treat these infections have not been developed. Surgical intervention is currently the standard of care for pre-invasive cervical lesions and overtreatment out of concern for progression or underlying high grade lesions is found frequently. Thus, effective and inexpensive novel topical and systemic pharmaceutical treatment options would be of great medical and financial benefit.It has been extensively reported that the natural trioxane artemisinin, the active principle of the Chinese medical herb Artemisia annua and its monomeric derivatives distinguish themselves as a new generation of very effective antimalarials with fewer toxic side effects than any other antimalarial drug. Recently, certain artemisinin derivatives were shown to inhibit the growth of a limited number of cancer cell lines.Frequently, cervical cancer cells overexpress the transferrin receptor to increase their iron uptake. We hypothesized therefore that iron dependent antimalarials such as artemisinin might prove useful in treating HPV-infected or transformed cells. We were able to show that dihydroartemisinin (DHA) and artesunate display strong cytotoxic effects on HPV immortalized and transformed cervical cells in vitro with little effects on normal cervical epithelial cells. DHA induced cell death involved the formation of reactive oxygen species and the activation of the mitochondrial caspase pathway with resultant apoptosis. We furthermore show that DHA can inhibit papillomavirus induced mucosal tumor growth in animals.We have now designed two new very stable artemisinin derived trioxane dimers with considerably higher selective anticancer activity in vitro than monomeric artemisinin and its derivatives. Specifically, we demonstrate that these dimers



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express a rapid, dose dependent and more than 500-fold higher cytotoxic activity towards human cervical cancer cells than DHA, whereas normal cervical cells are virtually unaffected.Our findings suggest that artemisinin derivatives, in addition to their well studied antimalarial acivities, may be clinically useful as potent chemotherapeutic agents for the treatment of cervical cancer, its precursors and potentially other mucosal and epidermal tumors. The stability and hydrophobicity of these dimers make them excellent candidates for not only systemic but also topical (e.g. intravaginal) application, a route of administration which would also permit high dosaging without the risk of systemic side effects. Topical application to early cervical dysplasia could greatly simplify the treatment of such papillomavirus-related lesions, including those in immunocompromised patients.



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Session 2

Medicinal plants and immune related diseases

Recent progress in Echinacea researchRudolf Bauer (Austria)

Immunomodulatory alkylamides in Echinacea, what is their role?Jürg Gertsch (Switzerland)

Immunomodulatory, pharmacological and therapeutic properties of MenthaMarco Cosentino (Italy)



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RECENT PROGRESS IN ECHINACEA RESEARCH

Rudolf Bauer

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-University, Graz, Austria

Echinacea angustifolia DC., Echinacea pallida (Nutt.) Nutt. and Echinacea purpurea (L.) Moench are known as herbal immunomodulators. Traditionally,roots and aerial parts of these plants have been used to treat wounds as well as insect and snake bites. Today Echinacea is mainly used for infections of the upper respiratory tract. In order to obtain consistent quality of batches, analysis of the relevant constituents is necessary. Alkamides and caffeic acid derivatives can be analysed by HPLC, determination of polysaccharides/glycoproteins is possible by specific ELISA methods. Recent investigations have shown that caffeic acid derivatives of Echinacea are rapidly oxidised by a polyphenol oxidase present in the plant material. The pharmacokinetic properties of Echinacea alkamides have recently been studied using ion trap ESI LC-MS/MS. The studies have demonstrated that Echinacea alkamides are detectable in human blood already 10 minutes after oral application.There is evidence that alkamide containing Echinacea preparations trigger effects on the pro-inflammatory cytokine TNF-α and chemokine IL-8 from a recent ex vivo study. Gertsch et al. could demonstrate modulation of TNF-α gene expression and multiple signal transduction pathways for Echinacea alkamides and postulated a mechanism related to cannabinoid receptors. Because of the structural similarity of Echinacea alkamides and anandamide, the endogenous ligand of CB receptors, receptor binding studies with alkamides to rodent CB1 and CB2 receptors were conducted in parallel. By Woelkart et al. it could be demonstrated that Echinacea alkamides have in fact high affinity to CB receptors. More evidence of CB2-receptor binding has recently been demonstrated by Raduner et al..Although the outcome of clinical studies with Echinacea preparations is not consistent, and the evidence of activity differs from product to product, a recent Cochrane review came to the conclusion, that especially preparations based on the aerial parts of Echinacea purpurea have some evidence to be effective. According to a meta-analysis with three selected experimental infection studies, the likelihood of experiencing a clinical cold was 55 % higher with placebo than with Echinacea.



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IMMUNOMODULATORY ALKYLAMIDES IN ECHINACEA: WHAT IS THEIR ROLE?

Jürg Gertsch, MSc, PhD

ETH Zürich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Str. 10, CH-8093 Zürich, Switzerland

Purple coneflower (Echinacea purpurea and E. angustifolia) preparations are widely used herbal medicines for the treatment of the common cold and upper respiratory infections. The estimated sales volume of Echinacea products worldwide is in the range of 800 million US dollars. Fatty acid N-alkyl amides (alkylamides) are a class of bioavailable lipophilic compounds generally found in Echinacea species. There is increasing evidence that N-isobutyl amides inhibit acute inflammation and might contribute to the beneficial effects reported for Echinacea. We have recently shown that two major alkylamides from Echinacea, dodeca-2E,4E-dienoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide potently interact with the cannabinoid type-2 (CB2) receptor and mediate anti-inflammatory effects in different cellular systems. Significant CB2 receptor-specific cellular signaling can be achieved with low nanomolar (nM) concentrations of CB2-active alkylamides in vitro, and there is preliminary evidence for superadditive effects in complex mixtures. Based on this relatively well-defined molecular mechanism, attempts in our laboratory to standardize Echinacea extracts and commercial products for their CB2 effect are presented. It is shown that some alkylamides tend to self-assemble into micelles and supermicelles, which may, at least in part, explain the effect differences between the isolated and extract-based alkylamides. A critical analysis of the potential pharmacological role of this interesting class of natural products in Echinaceaproducts is provided.



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IMMUNOMODULATORY, PHARMACOLOGICAL AND THERAPEUTIC PROPERTIES OF MENTHA

Elena Carcano, Raffaella Bombelli, Alessandra Luini, Franca Marino, Sergio Lecchini, Marco Cosentino

Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Varese, Italy

Mentha plants have been used and valued as aromatic herbs for thousands of years. “Mint” is the common name of approximately 25 perennial species of thegenus Mentha of the Lamiaceae family. Mentha species, in particular peppermint, are a source of essential oils which are valuable neutral products used as raw materials in many fields, including perfumes, cosmetics, aromatherapy, phytotherapy, spices and in nutrition. Both the essential oils and extracts isolated from members of the genus Mentha are used for medical purpose in herbal and pharmaceutical preparations and in traditional medicine. Mentha extracts and essential oils are a rich source of polyphenolic compounds, monoterpenes and, to a less extent, sesquiterpenes. Mentha species are considered aromatic, stimulant and carminative. In particular peppermint is commonly used as a herbal agent in folk medicine in the treatment of loss of appetite, common cold, bronchitis, sinusitis, fever, vomiting, and indigestion. Mentha essential oils are also used to allaying nausea and flatulence, and as promoter of gastric secretion. All these uses however still rely on traditional knowledge and neither clinical efficacy nor the cellular and molecular mechanisms involved have been examined in depth, so far. Significant attention on the contrary has been devoted to the spasmolytic, antibacterial and antifungal activities of Mentha essential oils, as well as to the antioxidant and anti-tumor properties of some constituents of Mentha, like eugenol and rosmarinic acid. The role of some components of Mentha in alleviating inflammatory diseases including arthritis, rheumatism, and acne skin allergy is also well documented at least in some experimental models. Essential oils of Mentha are used as anti-inflammatory remedies in aromatherapy, as well, although in this case their possible mechanism(s) of action is still matter of speculation. Mentha preparations are usually well tolerated, although some constituents of essential oils (namely, pulegone, menthone and menthofuran) may be toxic under certain conditions. Mentha preparations and extracts have a well-established role in folk medicine, however up to now only a few of their pharmacological properties have been investigated in depth. In particular, existing evidence regarding their antibacterial, antifungal, antioxidant, antitumor and immunomodulatory activities seems promising, but it still needs further support from both mechanistic studies as well as from well-conducted clinical trials.



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Session 3

Medicinal plants and neurodegenerative diseases

Neuroprotective activity of curcumin in in vitro models of neurodegeneration. Its potential as a therapeutic tool against neurodegenerationFiorella Malchiodi-Albedi (Italy)

Mechanisms of action of polyphenols in biological systems: effects on intracellular redox balanceRoberta Masella (Italy)

Dissection of the cell signalling pathway implicated in the mechanisms of neuroprotection afforded by the essential oil of bergamot against excitotoxic neuronal deathM. Tiziana Corasaniti, Simona Maida, Vincenza Fratto, Michele Navarra and Giacinto Bagetta (Italy)

From medicinal plants to multifunctional therapy for neurodegenerative diseases: pharmacodynamic and pharmacokinetic challengesPierre-Alain Carrupt (Switzerland)

The neuroprotective capacity of some Asteraceae and their polyphenol moleculesFederico Dajas (Uruguay)



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NEUROPROTECTIVE ACTIVITY OF CURCUMIN IN IN VITRO MODELS OF NEURODEGENERATION. ITS POTENTIAL AS A THERAPEUTIC TOOL AGAINST NEURODEGENERATION

F. Malchiodi-Albedi1, A. Matteucci,2 C. Frank,3 M.R. Domenici,3 M. Balduzzi,4 S. Paradisi1, G. Carnovale-Scalzo5, G. Scorcia5

Departments of 1Cell Biology and Neuroscience and 3Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome; 2G.B. Bietti Foundation for Ophthalmology (I.R.C.C.S.), Via Livenza 3, Rome, Italy;4UTS Biotechnologies, Protection of Health and Ecosystems, ENEA, Via Braccianese, Rome, Italy; 5Eye Clinic, University of Catanzaro “Magna Graecia”, Catanzaro, Italy

Curcumin, the major component of the spice turmeric (Curcuma longa), is widely used as food additive and herbal medicine in Asia. Investigations on the biological activity of curcumin have demonstrated a pletora of pharmacological properties, including anticarcinogenic, antiinflammatory and antioxidant effects. Since many pathological conditions of the central nervous system, such as AD, ischemia, degenerative retinopathy, involve, among others, chronic inflammatory response and oxidative stress, curcumin has been considered a promising compound potentially useful for the treatment of such disorders. Here curcumin was tested as protective agent against a well-described in vitro models of neurodegeneration: overstimulation of N-methyl-D-aspartate (NMDA) receptors (excitotoxicity) in rat retinal cultures. Incubation of retinal cell cultures for 24h with 1 mM NMDA induced an evident neuronal cell damage, estimated as decrease of cell viability by the assay of the conversion of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and increase in apoptosis, evaluated by the terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay and Hoechst stain. A 24h-treatment with curcumin reverted both NMDA-mediated decrease in cell viability and increase in apoptosis. By immunostaining GABAergic neurons (amacrine and horizontal cells) with anti-GABA and photoreceptors with anti-rhodopsin and evidencing apoptotic nuclei with Hoechst, we found that photoreceptors were almost unaffected by NMDA, while the GABAergic component showed a marked increase in apoptosis, which was reduced by curcumin pre-treatment. By performing optical fluorimetric recordings with fura-2AM, we evaluated the effects of curcumin pre-treatment in [Ca2+]i rise, following NMDA administration. We found that pre-treatment with curcumin reduced NMDA-mediated [Ca2+] i rise, suggesting that the observed neuroprotection can be



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correlated to a decrease in the NMDA-induced [Ca2+] i influx. We then characterized the time-dependency of curcumin neuroprotection. Interestingly, we found that neuroprotection could be evidenced for pre-treatment longer than 6h and increased to 24h while shorter incubations before NMDA treatment were ineffective. Similar results were observed in terms of [Ca2+] i recordings. The long incubation required to induce curcumin neuroprotection suggested that protein synthesis was involved in the mechanism. In fact, incubation with cycloheximide, which inhibits protein synthesis, abolished curcumin neuroprotection.

Curcumin has a well-known activity as kinase inhibitor. The phosphorylation state of the different subunits of the NMDA receptor regulates its activity and has a role in excitotoxic cell damage, since activation of protein kinases or inhibition protein phosphatases increase neuronal responses to glutamate receptor agonists. We therefore checked the phosphorylation state of the receptor. We did find a decrease in NR1 phosphorylation; however, in a time-course experiments, we observed that the decrease in phospho-NR1 was higher at 6h, but then increased again, so that neuroprotection and dephosphorylation peaks did not coincide. We then analyzed curcumin effect on NR2A and NR2B expression, in terms of immunocytochemistry and western blotting. While NR2B was not apparently affected, NR2A expression increased after 6 and 24 h of curcumin pre-treatment. Since NR2B is more involved in excitotoxicity, an increased expression of NR2A could explain the neuroprotecive effect induced by curcumin.

These results enlighten a new pharmacological action of the plant extract, possibly mediated by a modulation of NMDA receptor activity.



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MECHANISMS OF ACTION OF POLYPHENOLS IN BIOLOGICAL SYSTEMS: EFFECTS ON INTRACELLULAR REDOX BALANCE

Roberta Masella, Claudio Giovannini

National Centre for Food Quality and Risk Assessment, Istituto Superiore di Sanità, Rome, Italy

Oxidative stress, consequent to an intracellular redox imbalance, determines cellular damage with functional alterations of the involved tissue and has been associated with many forms of programmed cell death since free radicals can induce the apoptotic process. Thus oxidative stress is considered to play a pivotal role in the pathogenesis of aging and several degenerative diseases. To cope with an excess of free radicals, humans have developed enzymatic and non-enzymatic endogenous antioxidant/anticytotoxic defences in order to maintain redox homeostasis. Exogenous antioxidants are supplied with the diet. Polyphenols compounds, contained in fruit and vegetables, show in vitro strong antioxidant capacities since they act as chain breakers or radical scavengers. Thus their biological activity have been mainly attributed to the antioxidant properties. However, accumulating evidence indicates that polyphenols exhibit several additional properties in complex biological systems. They can modulate enzymatic activities, interact with signal transduction pathways and cell receptors, and activate antioxidant/anticytotoxic endogenous defence systems. We have studied the protective effects exerted by different phenolic compounds on the alteration of the redox balance and the related cytotoxic events, induced by oxidized low density lipoprotein (oxLDL) in cellular models. We have demonstrated that polyphenols counteract the inhibition of cell proliferation and the apoptosis induced by oxLDL likely through the alteration of intracellular redox balance leading to free radicals accumulation in Caco-2 cells. The protective effects are associated with the improvement of endogenous defences, in particular to the increase of glutathione (GSH), the major non-enzymatic regulator of intracellular redox homeostasis. Glutathione can directly scavenge free radicals or act as a substrate for enzymes being oxidised to glutathione disulfide (GSSG). GSH depletion, occurred during oxidative stress, is counteracted by the suitable functioning of glutathione-related enzymes, glutathione peroxidase (GPx) and glutathione transferase (GST), involved in the antioxidant/detoxifying activity, glutathione reductase (GRed), responsible for the back-reduction of GSSH to GSH and γ-glutamylcysteine synthetase (γ-GCS), responsible for GSH de novo.



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synthesis. Several experimental data indicate the tight interrelation betweenendogenous antioxidants and dietary polyphenols. We have demonstrated that oleuropein and protocatechuic acid contained in extra virgin olive oil remarkably increase GRed and mainly GPx activities, in murine macrophage-like cells J774 A.1. The increase in enzyme activities is likely dependent on the up-regulation of their mRNA induced by the polyphenols. In the same experimental model we have also shown that polyphenols are able to strongly induce the expression of the antiapoptotic/antioxidant Bcl-2 protein, belonging to Bcl-2 family proteins. The strengthening of the endogenous antioxidant defence system and the coordination of cell response to redox imbalance by polyphenols is obtained at least in part through the activation of Antioxidant Responsive Elements (ARE) found in the promoters of many inducible genes. The polyphenols influence directly ARE activity or the pathways that regulate ARE activation, in particular the Nrf2-Keap1 pathway.



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Dissection of the cell signalling pathway implicated in the mechanisms of neuroprotection afforded by the essential oil of bergamot against excitotoxic neuronal death

M. Tiziana Corasaniti1,2, Simona Maida1, Vincenza Fratto1, Michele Navarra3 and Giacinto Bagetta4

1Department of Pharmacobiological Sciences, University “Magna Graecia” of Catanzaro, 88100 Catanzaro, Italy; 2 Mondino-Tor Vergata Center fo Experimental Neuropharmacology, University of Rome Tor Vergata, 00133 Rome, Italy; 3Department of Pharmacobiology, University of Messina, 98168 Messina, Italy; 3Department of Pharmacobiology, UCADH Center of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Via P. Bucci, 87036 Arcavacata di Rende (CS), Italy.

The essential oil of bergamot (BEO) comprises a volatile fraction (93-96% of total) and a non volatile fraction (4-7% of total) containing waxes, polymethoxylated flavones, coumarins and psoralens i.e. bergapten and bergamottine (1). The effects of this phytocomplex on excitotoxic neuronal damage were investigated both in vitro and in vivo. The study was performed in human SH-SY5Y neuroblastoma cells exposed to NMDA and in rats subjected to focal cerebral ischemia.NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, and this was preceded by a significant accumulation of intracellular reactive oxygen species (ROS) and by a fast activation of the calcium activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream glycogen synthase kinase-3β (GSK-3β). BEO (0.0005-0.01%) concentration-dependently reduced death of SH-SY5Y cells caused by NMDA. In addition to prevent ROS accumulation and activation of calpain, BEO (0.01%) counteracted deactivation of Akt and the consequent activation of GSK-3β induced by NMDA. Results obtained by using specific fractions of BEO, suggest that monoterpene hydrocarbons are responsible for neuroprotection afforded by BEO against NMDA-induced cell death. Interestingly, intraperitoneal (i.p.) administration of BEO (0.5 ml kg-1, given 1 hour before middle cerebral artery occlusion, MCAo) significantly reduced infarct size after focal cerebral ischemia in rat and prevented reduction of phospho-Akt and phospho-GSK-3β levels in the ischemic cortex, without affecting Akt and GSK-3β protein expression.In conclusion, the data demonstrate that BEO reduces neuronal damage caused in



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vitro and in vivo by excitotoxic stimuli and that neuroprotection is associated with prevention of injury-induced decrease of pospho-Akt and phospho-GSK-3β levels.

Financial support from MiUR (PRIN 2004055288_1) and Calabria Region (POR 2000-2006) to CMT is gratefully acknowledged.

1) Dugo P., Mondello L., Dugo L., Stancanelli R., Dugo G. (2000) J. Pharm. Biomed. Anal. 24: 147-150.



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FROM MEDICINAL PLANTS TO MULTIFUNCTIONAL THERAPY FOR NEURODEGENERATIVE DISEASES: PHARMACODYNAMIC AND PHARMACOKINETIC CHALLENGES

Pierre-Alain Carrupt, Marianne Reist, Sophie Martel

LCT- Pharmacochemistry, School of pharmaceutical sciences, University of Geneva, University of Lausanne, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

Intermolecular interactions between a well-defined chemical compound and only one biological target responsible for the therapeutic effect were widely used to develop drug compounds. Recent efforts in pharmaceutical research focus on new therapeutic strategies dealing with multifunctional pharmaceutical preparations able to interact efficiently with several targets selected for a given disease in order to lower compliance problems.Without doubt, medicinal plants offer numerous possibilities to identify and select multifunctional preparations. However, plant extracts are most often a complex mixture of several monofunctional compounds or possibly multifunctional compounds. Some rational screening strategies will be discussed in order to characterize the multifunctional pharmacodynamic profile of plant extracts. They also allow to identify multifunctional natural compounds able to solve problems associated with multi-drug therapy such as different pharmacokinetic properties and to lower risks of potential drug–drug interactions. Some interesting multifunctional hits in the field of aging related neurodegenerative diseases will be presented as well as further pharmacomodulation and focused libraries preparation guided by in silico optimization of pharmacodynamic and pharamcokinetics profiles.



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THE NEUROPROTECTIVE CAPACITY OF SOME ASTERACEAE AND THEIR POLYPHENOL MOLECULES

Dajas F., Rivera F., Blasina F., Echeverri C., Arredondo F., Vaamonde L.

Clemente Estable Institute, Avda Italia 3318, 11600, Montevideo, Uruguay.

Brain diseases have been linked to oxidative stress and epidemiological studies indicate that dietary antioxidants can modify their incidence. Consequently, herbs with a high concentration of flavonoids – recognized antioxidant molecules - such as those of the Asteraceae family are of special interest to explore in their brain protective capacity.Achyrocline satureioides (AS, “Marcela”) and Happlopapus parvifolius (HP, “Bailahuen”) taken as representatives of the family have shown a high antioxidant, cyto and neuroprotective capacity (through antiapoptotic and antinecrotic mechanisms) for cells and neurons in culture. These effects appear to be due to their high content in a group of aglycone polyphenols structurally related to quercetin such as myricetin, fisetin or luteolin. Though other plant extracts such as Ginkgo biloba and Epilobium parviflorum also show high antioxidant activity, only AS and HP were cytoprotective. Polyphenols of AS such as quercetin also protected the brain against cell death induced by permanent focal ischemia in rats as well as severe hypoxia in newborn piglets.Beyond their scavenger activity, the intracellular targets of flavonoids are multiple, from homeostasis of calcium to involvement with key intracellular signalling cascades. The flavonoid molecule can become a template for compounds therapeutically active in stroke or dementia and plant extracts with high free flavonoid content can be of value in the prevention of brain diseases.



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Session 4

Medicinal plants and safety

HET-CAM-assay - a non-animal approach to assess mucous membrane irritation of essential oilsJürgen Reichling (Germany)

Molecular mechanism of cancer caused by Aristolochia plantsHeinz Schmeiser (Germany)



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HET-CAM-ASSAY– A NON-ANIMAL APPROACH TO ASSESS MUCOUS MEMBRANE IRRITATION OF ESSENTIAL OILS AND SINGLE OIL COMPOUNDS

Jürgen Reichling, Jürgen Schneele

University of Heidelberg, Institute of Pharmacy and Molecular Biotechnology, Department of Biology, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

Background: The Rabbit-Eye-Draize-Test is the standard procedure to assess eye irritation potential of substances: label R41, strong irritants; label R36, low irritants. Essential oils and their components which are often ingredients of cosmetic products are known as low irritants. On the other hand, in Germany, the Draize-Test is forbidden to assess the irritation potential of a new developped cosmetic product.Objectives: Development of a modified HET-CAM-Assay to predict the putative irritaion potential of essential oils and their single components in vitro.Methods: Test substances in native olive oil were applied to the chick embryo chorioallantoic membrane (CAM). The irritation threshold concentration (ITC) was determined as the lowest concentration of the test substance that causes the end point of haemorrhage on the CAM´s blood vessels within 5 min. SDS was used as positive control and native olive oil as negative control.Results: The results obtained reveal that the modified HET-CAM-Assay protocol is a useful tool to rank essential oils and their single components according to their irritation potential (ITC values). Clove Oil and rose oil were found to be the most irritant essential oils tested. For essential oil single components a structure-irritation-relationship was identified. Conclusion: The new HET-CAM-Assay protocol allows evaluating and ranking the irritation potential of essential oils and their single components according to their ITC values. Comparing the ITC values obtained with manufacturer labels for relevant irritation (R-36; risk indication: irritating to eyes) revealed its in vivorelevance.



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MOLECULAR MECHANISM OF CANCER CAUSED BY ARISTOLOCHIA PLANTS

Heinz H. Schmeiser

Division of Molecular Toxicology, German Cancer Research Centre, 69120 Heidelberg. [email protected]

The use of a herbal weight-loss product, containing Aristolochia species has been associated with the development of a novel nephropathy, and urothelial cancer (Arlt et al 2002). The observed nephropathy and urothelial carcinoma has been traced to the ingestion of aristolochic acid (AA) and is now called aristolochic acid nephropathy (AAN). The major components of the plant extract AA, aristolochic acid I (AAI) and aristolochic acid II (AAII), both are nitrophenanthrene carboxylic acids, which are genotoxic mutagens forming DNA adducts after metabolic activation. Several mammalian enzymes have been shown capable to activate both AAI and AAII in vitro and in cells. The activating metabolism has been elucidated and is consistent with the formation of a cyclic nitreniumion with delocalised charge leading to the preferential formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine and deoxyguanosine. The predominantly formed DNA adduct 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) in vivo was detected in AAN patients unambiguously proofing the exposure to AA. dA-AAI the most persistent adduct in target tissue and therefore detectable in urothelial tissue from AAN patients 10 years after they stopped taking the weight-loss regimen is a mutagenic lesion leading to AT→TA transversions in vitro. This transversion mutation is found in high frequency in codon 61 of the H-ras oncogene in tumours of rodents induced by AAI, suggesting that dA-AAI might be the critical lesion in the carcinogenic process in rodents (Arlt et al 2002). Interestingly, in one AAN patient available for analysis the identical AT→TA transversion mutation was found in the p53 gene in urothelial tumour cells. In contrast, the molecular mechanism of renal interstitial fibrosis in humans after chronic administration of AA remains to be explored. AA is a powerful nephrotoxic and carcinogenic substance with an extremely short latency period not only in animals but also in humans. In particular the highly similar metabolic pathway of activation and resultant DNA adducts of AA which are detectable by the 32P-postlabeling method, allows the extrapolation of carcinogenesis data from laboratory animals to the human situation. These findings draw one of the strongest links yet between use of a herbal product and cancer in humans. Therefore, all botanical-containing products known or suspected of containing AA should be banned from the market worldwide.References: Arlt, V.M., Stiborova, M., Schmeiser, H.H. 2002 Mutagenesis 17, 265-77



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Session 5

Methodological issues in clinical trialswith medicinal plants

Phytomedicine IS scientific medicine: What’s next?Heinrich Walt (Switzerland)

From standard Tanacetum Parthenium extract to a stabilized derivative of parthenolide: studies in animal models of painCristina Tassorelli, Paolo Morazzoni (Italy)

Methodology and reporting of clinical trials of herbal medicine interventionsJoel Gagnier (Canada)



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PHYTOMEDICINE IS SCIENTIFIC MEDICINE: WHAT’S NEXT?

Heinrich Walt

Research Division of Gynecology, University Hospital, Zurich, Switzerland

Methodological communication was not a major problem during the early days of medicine. Treatment modalities were based on a gift from mother nature: The plants and extracts thereof. They were widely known for centuries. Thus, Phytomedicine was Scientific Medicine. Starting with the 19th century, after establishing clinical chemistry, followed later on by many new developments based e.g. on biomedical engineering, physics and molecular medicine, new avenues of medical research were paved. These developments had their own powerful way, leaving Phytomedicine separated, and one might argue that during this phase Phytomedicine was not Scientific Medicine. Besides the groups that kept traditional medicine (including Phytomedicine) alive, there were more and more scientists of modern education that found interesting aspects in studying the traditional discipline by use of the most recent methodology. Chemical formulas and details about molecules that are fundamental for both modern Phyto- and Scientific Medicine. These researchers have published in peer reviewed journals, thus initiating the way to bring together these medical disciplines that were separated for such a long time. This indicates that Phytomedicine is Scientific Medicine. Our round table discussion will start with a short presentation of an ongoing project in our institution in order to better illustrate the last sentence. According to the mission of this meeting, we will focus on improving communication and we will address actual problems. We hope that they can be resolved with interdisciplinary help: more focused research, enclosure of interested chemical industry and help of the politicians. Your valuable contribution during the round table will elucidate further topics to be listed.



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FROM STANDARD TANACETUM PARTHENIUM EXTRACT TO A STABILIZED DERIVATIVE OF PARTHENOLIDE: STUDIES IN ANIMAL MODELS OF PAIN

Cristina Tassorelli1, Paolo Morazzoni2, Rosaria Greco1, Antonella Riva2, Giorgio Sandrini1, Giuseppe Nappi1,3

1Laboratory of Pathophysiology of Integrative Autonomic Systems, University Center for the Study of Adaptive Disorders and Headache (UCADH), IRCCS ‘C. Mondino Institute of Neurology’ Foundation, Pavia, Italy - 2Indena S.p.A., Milan, Italy - 3Department of Clinical Neurology and Otorhinolaryngology, University La Sapienza, Rome, Italy

Parthenolide is a chemical component and, potentially, the principal active ingredient of Tanacetum Parthenium (TP) - an extract that has proven to some extent effective in the treatment of painful conditions, including migraine. In order to test the hypothesis that parthenolide is the active principle of TP as concerns its potential antimigraine activity, we evaluated the effect of extracts of TP with different contents in parthenolide and purified parthenolide (Pp) in a well-known animal model of migraine, based on the administration of nitroglycerin (NTG) in the rat. The findings obtained demonstrated that only the extract enriched in parthenolide significantly interfered with the neurochemical mechanisms that are relevant for migraine in specific nociceptive structures located in the medulla, i.e. nucleus trigeminalis caudalis (NTC). A more widespread effect – extended to higher centers - was observed with Pp, which indeed inhibited NTG-induced neuronal activation in several brain nuclei besides NTC. As a subsequent step, Pp was stabilized through a cysteinylation reaction, in order to prevent the decrease of its activity over time. The antinociceptive activity of the cysteinyl derivative was then demonstrated in models of tonic and phasic pain. Through this articulated and virtuous process of “purification-testing in models-adaptation-retesting in models” we came to the conclusion that parthenolide is the active principle of TP and that its stable derivative may be useful in the treatment of migraine and other painful conditions.



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METHODOLOGY AND REPORTING OF CLINICAL TRIALS OF HERBAL MEDICINE INTERVENTIONS

Joel J. Gagnier

Post-Graduate Fellow, Faculty of Medicine, University of Toronto, CanadaSenior Science Officer, Jamieson Laboratories Ltd, Windsor, Ontario, Canada

It has been argued that the best evidence for clinical interventions stems from systematic reviews and randomized controlled trials (RCTs). But, poorly reported and performed studies can give a misleading impression of efficacy and questionable clinical applications of trial results. Specifically, poor methods may bias estimates of treatment effect and poor reporting may lead to unreliable and invalid estimates of bias. Herbal medicine trials are poorly reported. Several studies have found that RCTs of herbal medicine interventions reported less than half of the information suggested in the CONSORT statement. This appears to be similar for trials of conventional interventions. Without adequate reporting, it is difficult to asses the influence of bias in RCTs. There are unique aspects of herbal medicine RCTs that require reporting, beyond the generic information suggested in CONSORT. Recently, we developed and published guidelines for reporting RCTs of herbal medicine interventions. Also, several studies have explored the methodological quality of herbal medicines trials. The quality is generally quite good. Overall, RCTs of herbal interventions can be improved. Adequate methods will allow for accurate estimates of treatment effect and clear reporting will contribute to accurate attribution of treatment effects to specific interventions and allow for clinical application and repetition of RCTs.



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Poster Session(presenting Authors in alphabetical order)



EFFECT OF WITHANIA SOMNIFERA EXTRACTS ON DENDRITIC SPINES LOSS IN THE RAT NUCLEUS ACCUMBENS DURING OPIATE WITHDRAWALAcquas E., Spiga S., Vinci S., Ibba F., Kasture S.B.

A COMPARATIVE STUDY OF RADIO-PROTECTION BY CURCUMA LONGA, TINOSPORA CORDIFOLIA & ZIZYPHUS MAURITIANA.Adhvaryu M.R., Vakharia B.C., Reddy M.N.

IN VITRO ANTICANCER ACTIVITY OF E. PALLIDA POLYACETYLENESAdinolfi B., Chicca A., Pellati F., Benvenuti S., Massarelli I., Martinotti E., Nieri P.

BIOASSAY-DIRECTED ISOLATION OF HYPOTENSIVE ALKALOID FROM HOLARRHENA PUBESCENSAftab K., Usmani S.B., Begum S., Siddiqui B.S.

7-HYDROXYMATAIRESINOL POTASSIUM ACETATE (HMR/lignanTM) FROM NORWAY SPRUCE (PICEA ABIES) KNOTS AND ITS ACTIVE METABOLITE ENTEROLACTONE EXERT ESTROGENIC EFFECTS IN THE IN MCF-7 CELL MODELBombelli R., Marino F., Ferrari M., Rasini E., Luini A., Legnaro M., Delle Canne M.G., Luzzani M., Crema F., Paracchini S., Lecchini S., Cosentino M.

IMMUNOMODULATORY ACTIVITIES OF ACHYROCLINES SATUREIOIDES ('MARCELA'): AN IN VITRO STUDYBombelli R., Carcano E., Luini A., Marino F., Lecchini S., Cosentino M., Dajas F.

ANTIOXIDANT AND CYTOPROTECTIVE PROPERTIES OF INFUSIONS OF ACHILLEA MILLEFOLIUM SSP. COLLINABombelli R., Giorgi A., Luini A., Licheri G., Carcano E., Marino F., Cosentino M., Cocucci M., Lecchini S.

P-01

P-02

P-03

P-04

P-05

P-06

P-07

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FREE RADICAL SCAVENGING ACTIVITY AND EFFECTS ON THE OXIDATIVE METABOLISM OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE OF MENTHA PIPERITA ESSENTIAL OILSBombelli R., Luini A., Carcano E., Marino F., Colombo M.L., Conti A., Cosentino M., Lecchini S.

IMMUNOMODULATORY EFFECTS OF DIFFERENT MENTHA PIPERITAVARIETIES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLSCarcano E., Cosentino M., Bombelli R., Luini A., Marino F., Colombo M.L., Conti A., Lecchini S.

IN VITRO ANTI -TUMORAL ACTIVITY OF PISTACIA LENTISCUSPHYTOCOMPLEXESBuriani A., Fortinguerra S., Signorelli A., Innocenti G., Dall’Acqua S., Barbera M., Caputo A., Carrara M.

ANTIOXIDANT CAPACITY AND CHEMICAL CHARACTERIZATION OF HONEY FROM HERBS PRODUCED IN MOUNTAIN AREASGiorgi A., Madeo M., Francescato P., Cairoli P., Speranza G.

ANTI-INFLAMMATORY EFFECTS OF A TRADITIONAL KOREAN HERBAL MEDICINE, SCUTELLARIAE RADIXHam I., Ko Y., Lee J., Yang G., Jun K., Park J., Park C., Choi H.

HEALING EFFECTS OF ALCHORNEA TRIPLINERVIA ON ENHANCE THE COX2 AND PROSTAGLANDIN E2 EXPRESSIONLima Z.P., Calvo T.R., Silva E.F., Pellizzon C.H., Vilegas W., Brito A.R.M.S.,Hiruma-Lima C.A.

DAVILLA NITIDA: ROLES OF ENDOGENOUS SULPHYDRYLS AND NO IN GASTRIC PROTECTION AGAINST LESIONS INDUCED BY ETHANOLKushima H., Clenilson M. Rodrigues C.M., Rocha L.R.M., Cola M., Brito A.R.M.S., Rinaldo D., Santos L.C., Villegas W., Hiruma-Lima C.A.

GASTRIC ULCER HEALING PROMOTED BY FLAVONOIDS AND TANNINS FROM THE MEDICINAL PLANT MOURIRI PUSA GARDN.(MELASTOMATACEAE)de Paula Vasconcelos P.C., Andreo M., Hiruma-Lima C.A., Vilegas W., Pellizzon C.H.



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FENNEL TEA IN INFANCY – A BENEFIT-RISK-EVALUATIONIten F., Saller R.

PRECLINICAL EVALUATION OF STANDARDIZED EXTRACT OF MUCUNA PRURIENS IN ANIMAL MODELS OF PARKINSON’S DISEASEKasture S., Pontis S., Simola N., Pinna A., Spina L., Longoni R., Micaela M.

ANTIOXIDANT EFFECT OF THE ETHYL ACETATE EXTRACT OF TEUCRIUM POLIUM L. ON CCL4 INDUCED DAMAGE IN RAT’S LIVERKadifkova Panovska T., Kulevanova S., Gjorgoski I., Bogdanova M., Petrushevska G.

A NEW MODEL FOR STUDY OF HYPO- AND HYPERTENSIVE EFFECTS IN OVOMakarova M., Selesneva A., Makarov V.

AN UPDATED SYSTEMATIC REVIEW WITH META-ANALYSIS OF THE CLINICAL EVIDENCE OF SILYMARINSaller R., Brignoli R., Melzer J., Meier R.

INFLUENCE OF ISOFLAVONES ON THE PROTEIN EXPRESSION IN HUMAN LIVER (Hep G2) AND BREAST (MCF 7) CANCER CELLS – A PROTEOMIC APPROACHPakalapati G., Wink M.

PHYTOECDYSTEROID COMPOSITION OF MICROSORUM SPECIES (POLYPODIACEAE) OF FRENCH POLYNESIAHo R., Teai T., Loquet D., Bianchini J.-P., Girault J.-P., Lafont R., Raharivelomanana P.

MEDICINAL PLANTS FROM FRENCH POLYNESIA: EVALUATION OF THEIR FREE RADICAL SCAVENGING ACTIVITYLeu T., Soulet S., Loquet D., Meijer L., Raharivelomanana P.

FUCAN SULFATE DIRECTLY INHIBIT THE ACTIVITY OF SERIN PROTEINASESRepice C., Graziadio B., Distaso M., Iacobelli M., Echart C.

HET-CAM ASSAY FOR VASODILATATION EFFECT OF NANOSYSTEM WITH TAXIFOLIN Pozharitskaya O., Karlina M., Shikov A., Makarova M.N., Tikhonov V.P.



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ANTIULCER AND HEALING PROPERTIES FROM TWO VERNONIA POLYANTHES LESS. FRACTIONSBarbastefano V., Cola-Miranda M., Luiz-Ferreira A., Farias-Silva E., Takayama C., Rinaldo D., Hiruma-Lima C.A., Vilegas W., Souza Brito A.R.M.

EFFECT OF THE INDIGO FROM INDIGOFERA TRUXILLENSIS ON COX-1 EXPRESSION IN THE GASTRIC MUCOSA OF RATSFarias-Silva E., Calvo T.R., de Paula Michelatto D., Alves de Almeida A.C., Schwambach Vieira A., Barbastefano V., Cola-Miranda M., Luiz-Ferreira A., Hiruma-Lima C.A., Vilegas W., Souza Brito A.R.M.

BIOASSAY-GUIDED FRACTIONATION OF THAI MEDICINAL PLANTS USED TO TREAT CANCERTechatanawat I., Houghton P.J., Hylands P.J.

INHIBITORS OF ACETYLCHOLINESTERASE FROM GENTIANA AND PEUCEDANUM SPECIESUrbain A., Marston A., Hostettmann K.

ANTIDEPRESSIVE EFFECTS OF IGNATIA AMARA CONTAINING COMPLEX HOMEOPATHIC REMEDIESWasilewski B.W.

LIPID LOWERING ACTIVITY OF CITRI UNSHII PERICARPIUM IN HYPERLIPEMIC RATSYang G., Lee J., Jung E.-D., Ko Y., Lee B.-H., Ham I., Choi H.-Y.

CHARACTERISATION AND ANTIOXIDANT EFFECTS OF ESSENTIAL OILS OF MENTHA CITRATA AND MENTHA PIPERITAZanni M., Bombelli R., Marino F., Cosentino M., Conti A.

NONSTEROIDAL ANTI-INFLAMMATORY PROPERTIES OF SOME ALPINE MEDICINAL PLANTSMarcon N., Berthouzoz S., Grogg A.F., Zonnevijlle F.

POPULAR USES AND KNOWLEDGE ABOUT MEDICINAL PLANTS IN THE TICINO REGIONPoretti Lozano Becerra G., Conti A.



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P-01EFFECT OF WITHANIA SOMNIFERA EXTRACTS ON DENDRITIC SPINES LOSS IN THE RAT NUCLEUS ACCUMBENS DURING OPIATE WITHDRAWAL

E. Acquas1,2, S. Spiga3., S. Vinci1., F. Ibba1, S.B. Kasture1

1Department of Toxicology and 2Centre of Excellence on Neurobiology of Addiction, 3Department of Animal Biology and Ecology, University of Cagliari

Withania Somnifera (WS), a plant of the Solanaceae family, has been used in many indigenous cultures (Middle East, Africa and India) as a remedy for a number of conditions and diseases. Most recently a growing interest has focused on the potential neuritic regeneration and synaptic reconstruction properties of its C28 steroidal lactones derivatives (withanolides). Morphine withdrawal is associated with a number of electrophysiological, neurochemical and morphological changes thought an expression of a reduction of dopamine function in the ventral tegmental area and nucleus accumbens (NAc). These changes are paralleled by a reduction of spines' density of 2nd order dendrites of medium size spiny neurons (MSN) in the shell of the NAc. Aim of the present study was to determine whether morphine withdrawal-induced spines’ loss in the NAc could be affected by the administration of WS extracts. To this end rats were chronically treated with morphine or saline in combination with WS extracts and, at the completion of chronic treatment or upon spontaneous (1 day) or pharmacologically precipitated withdrawal, their brains were fixed in Golgi-Cox solution for microscopical examination. As expected both spontaneous and pharmacologically precipitated morphine withdrawal determined a dramatic reduction of spines’ density in MSN of the shell (but not core) of the NAc; pretreatment with WS extracts fully prevented both reductions of spines’ density. These findings, while indicating that pretreatment with WS extracts prevents the changes of structural plasticity induced by opiate withdrawal, suggest that withanolides might be beneficial on the behavioural consequences mediated by such synaptic rearrangements.



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P-02A COMPARATIVE STUDY OF RADIO-PROTECTION BY CURCUMA LONGA, TINOSPORA CORDIFOLIA & ZIZYPHUS MAURITIANA.

Meghna R. Adhvaryu, Bhasker C. Vakharia, M.N. Reddy.

Bapalal Vaidya Botanical Research Centre; Dept. Of Biosciences; Veer Narmad South Gujarat University; Surat-395007; INDIA. *E-Mail: [email protected]

Background: Synthetic radio-protective agents like aminothiols are toxic and FDA approved agent amfostine is no exception. Ocimum sanctum (OS) flavonoids, having very safe toxicological profile have shown comparable radio-protective potential in rodents. Likewise other medicinal herbs are likely to have similar activity. Objectives: To compare the radio-protective potential of Curcuma longa (CL) - a condiment with an active principle curcumin, Tinospora cordifolia (TC) - an ayurvedic herb used as Rasayana and Zizyphus mauritiana (ZM) – Indian Ber with Ocimum sanctum having similar efficacy with that of amfostine as control. Methods: Adult Swiss albino mice from random breed colony were divided in to 6 groups (n=9), control, radiotherapy (RT), OS + RT and three herb + RT groups respectively. All except control were exposed to whole body 2.0 Gy of gamma radiation in a teletherapy unit on day 7 of herb pretreatment (200mg/kg-b.w. orally by gavage). Chromosomal studies from the bone marrow of femur by routine metaphase preparation after colchicine treatment were done in 3 animals from each group at 24, 72 and 168 hours after exposure.Results: All three herbs showed significant radio-protective effects at 24 hrs. TC and ZM showed nearly similar activity while CL being the least effective. However the effects at 72 and 168 hrs showed highest protection by CL followed by ZM>TC>OS respectively suggesting that the well studied OS fares poorly at 72 and 168 hrs.Conclusions: All the 3 herbs and OS as well have radio-protective potential with different efficacies. As there is no single ideal radio-protective agent available, combination of multiple molecules having different mechanisms of action might prove superior.



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P-03IN VITRO ANTICANCER ACTIVITY OF E. PALLIDA POLYACETYLENES

Adinolfi B.1, Chicca A.1, Pellati F.2, Benvenuti S.2, Massarelli I.3, Martinotti E.1, Nieri

P.1

1Dep. Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, via Bonanno, 6, 56100, Pisa, Italy; 2Dep. Pharmaceutical Sciences, University of Modena and Reggio Emilia, via G. Campi, 183, 41100, Modena, Italy; 3Dep. Chemistry and Industrial Chemistry, University of Pisa, via Risorgimento 35, 56100, Pisa, Italy.

Root hexanic extracts from Echinacea angustifolia, pallida, and purpurea are cytotoxic on human pancreatic adenocarcinoma (MIA PaCa-2) and colon (COLO320) cancer cell lines and E. pallida is the most active species (1). This pharmacological evidence is in accordance with the different phytochemical profile of the E. pallida hexanic extract, rich of polyacetylenes which are almost absent in the other two species (containing mainly alkylamides) (2).Aim of this work was to investigate the effects of polyacetylenes, isolated by a bio-guided assay fractionation from the hexanic extract of E. pallida, on MIA PaCa-2 and COLO320 cancer cell lines. Cell viability was evaluated by the colorimetric WST-1 assay and expressed as % of control. Moreover, apoptotic cell death was assessed by an immunoenzimatic analysis of the cytosolic internucleosomal DNA enrichment and by the evaluation of caspase 3/7 activity. In the range 1-100 µM and for 24-72 h time-exposure a concentration- and time dependent cytotoxic effect was observed in both cell lines. A different cytotoxic profile among the constituents isolated was evident and a correlation between their effect and hydrophobicity was revealed. For some compounds an apoptotic cell death mechanism was also demonstrated. Moreover, IC50 values revealed COLO320 to be more sensitive than MIA PaCa-2 cells.In conclusion, our data show a cytotoxic activity of E.pallida polyacetylenes and suggest a possible contribution of these compounds in anticancer treatment.

(1) Chicca A. et al.(2007) J. Ethnopharmacol. 110: 148-153(2) Pellati F. et al. (2006) Phytochemistry 67: 1359-1364



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P-04BIOASSAY-DIRECTED ISOLATION OF HYPOTENSIVE ALKALOID FROM HOLARRHENA PUBESCENS

Khalid Aftab, S.B. Usmani, S. Begum and B.S. Siddiqui

*Department of Pharmacology & Therapeutics, Wah Medical College. Wah Cantt.H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.E-mail: [email protected] Fax # 9251-9314356

Holarrhena pubescens belongs to the family Apocynacea, commonly known as “kurchi” is highly reputed in traditional medicine as a remedy for amoebic dysentery and other intestinal ailment. Bioassay-directed fractionation of the ethanolic extract of Holarrhena pubescens resulted in the isolation of steroidal alkaloids i.e. Holamide and Pubscinine.Holamide showed a three proton doublet at 1.45 (J=6.56 Hz) and two AB doubles at 3.17 and 3.00 each for on proton (J=12.06 Hz) in the 1H NMR spectrum suggested that it belongs to conanine series of alkaloid (A class of compound with the steroid nucleus and a five members heterocyclic ring with nitrogen). In contrast Pubscinine showed one methyl at 1.28 while the doublet is missing a three proton singlet was observed at 2.28 due to a vinylic methyl indicated a double bond in the 18,20 – epimino ring of the conanine series of alkaloids.In anaesthetized rats, the Holamide and Pubscinine caused a fall in blood pressure in a dose-dependent manner. Pretreatment of animals Atropine completely abolished the hypotensive response of Acetycholine; whereas hypotensive effect of Holamide and Pubscinine were not modified by Atropine. Similarly Acetylcholine produced contractile effect in guinea-pig ileum, which was antagonized by atropine, however both (Holamide and Pubscinine) failed to produced any stimulant response on guinea-pig ileum. These data indicate that the steroidal alkaloids i.e. Holamide and Pubscinine from Holarrhena pubescens mediated hypotensive response through a mechanism different to that of Acetycholine.



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P-057-HYDROXYMATAIRESINOL POTASSIUM ACETATE (HMR/lignanTM) FROM NORWAY SPRUCE (PICEA ABIES) KNOTS AND ITS ACTIVE METABOLITE ENTEROLACTONE EXERT ESTROGENIC EFFECTS IN THE IN MCF-7 CELL MODEL

Raffaella Bombelli1, Franca Marino1, Marco Ferrari1, Emanuela Rasini1, Alessandra Luini1, Massimiliano Legnaro1, Marco Gioacchino Delle Canne2, Marcello Luzzani2, Francesca Crema3, Silvano Paracchini2, Sergio Lecchini1 , Marco Cosentino1

1Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Varese, Italy, 2Linnea SA, Riazzino (Locarno), Switzerland, and 3Department of Internal Medicine and Therapeutics, Clinical Pharmacology Unit, University of Pavia, Pavia, Italy

Several lines of evidence indicate that plant polyphenols belonging to the class of the lignans may possess anticancer, antioxidant, antimicrobial, anti-inflammatory and immunomodulatory activities. In particular, 7-hydroxymatairesinol (HMR/lignanTM, HMR) is a novel lignan which is converted to the mammalian lignan enterolactone (EL) by the gut microflora. In the present study, we investigated the estrogenic activity of HMR and of EL in comparison to estradiol (E2), by measuring their effects on growth and apoptotic markers in the human estrogen-sensitive cell line MCF-7. HMR, EL and E2 concentration-dependently increased the percentage of MCF-7 cells in the S phase of the cell cycle, with the following relative potencies: E2 ≅ EL >> HMR, and efficacies: E2 > HMR >> EL. Treatment of MCF-7 cells with either HMR, EL or E2 also increased the Bcl-2/Bax mRNA ratio. The effects of HMR and EL were reduced in the presence of the estrogen receptor (ER) antagonist tamoxifene. We conclude that both HMR and its metabolite EL are endowed with estrogenic activity, which is likely to be exerted through the contribution of ER-dependent pathways and to target the same intracellular mechanisms acted upon by E2. In comparison to E2, both HMR and EL showed however lower potencies and efficacies, suggesting a milder estrogenicity. The present results allow to conclude that dietary supplementation with HMR may result in a mild estrogenic activity, both directly and by providing a suitable source for endogenous EL.



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P-06IMMUNOMODULATORY ACTIVITIES OF ACHYROCLINES SATUREIOIDES('MARCELA'): AN IN VITRO STUDY

Raffaella Bombelli, Elena Carcano, Alessandra Luini, Franca Marino, Sergio Lecchini, Marco Cosentino, Federico Dajas§

Department of Clinical Medicine, Section of Clinical and Experimental Pharmacology, University of Insubria, Varese, Italy, and §Clemente Estable Institute, Montevideo, Uruguay

Achyrocline satureioides (AS) is a South American native medicinal herb known by the popular name of “Marcela”. Its infusion is widely utilized for the treatment of several digestive ailments, as an antinflammatory preparation, as a sedative, anti-atherosclerotic and for some nervous system disorders. Circumstantial evidence suggests that extracts of AS may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects AS infusions on various immune cell functions. Preparation of AS was performed by infusion of 1g of aerial parts in 50 ml of boiled water. The quality of the infusion was verified by the Folin-Ciocalteu method. Human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) were obtained by density-gradient centrifugation from venous blood of healthy donors. PBMCs were cultured for 48 h in standard conditions, alone or in the presence of the polyclonal mitogen phytohaemagglutinin (PHA) 10 µg/ml. PMNs were used in short-term (1 h) culture and were stimulated with fMLP 0.1 µM. Cell viability, assessed by the MTT and the LDH assay, was not affected by AS infusion up to 1:25 dilution. AS infusion concentration-dependently reduced PHA-induced proliferation of PBMCs down to complete inhibition at 1:50-1:25 dilutions. In the same concentration range, AS infusion also reduced PBMC production of interferon (IFN)-γ and of interleukin (IL)-4. The ratio IFN-γ/IL-4, which is usually taken as an index of the Th1/Th2 balance was not affected up to the 1:50 dilution, while with the 1:25 dilution it significantly increased with respect to control values. In human PMNs, AS infusion per se slightly increased reactive oxygen species (ROS) production but only at 1:50-1:25 dilutions. On the contrary fMLP-induced ROS production was effectively inhibited already at 1:1000 and nearly completely abolished at 1:200. The results of the present study clearly show that AS infusion may exert several immunomodulatory effects potentially relevant for its therapeutic use as an anti-inflammatory agent in many disease conditions. Further studies however are warranted to better characterize such effects and to assess their actual therapeutic relevance.



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P-07ANTIOXIDANT AND CYTOPROTECTIVE PROPERTIES OF INFUSIONS OF ACHILLEA MILLEFOLIUM SSP. COLLINA

R. Bombelli, A. Giorgi2 , A. Luini1, G. Licheri2, E. Carcano1, F. Marino1, M. Cosentino1, M. Cocucci2, S. Lecchini1

1Dipartimento di Medicina Clinica, Sezione di Farmacologia Sperimentale e Clinica, Università degli Studi dell’Insubria, Varese and 2Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Milano.

Introduction. Several lines of evidence indicate that antioxidants may help preventing heart diseases, cancer, aging and neurodegenerative diseases. Many medicinal plants contain large amounts of antioxidants. Among these plants, Achillea millefolium L. species have a wide traditional practice. Objective. The aim of this study was to evaluate the antioxidant and cytoprotective properties of infusions from leaves and inflorescences of Achillea millefolium ssp collina B and the relationship with their total phenolic content. Methods. The infusions were prepared by extracting aliquots of 0.05 g ofinflorescences or leaves with 10 mL of boiling water for 5 min. Antioxidant activity was determined with DPPH reagent. Oxidative damage was induced in PC12 cells cultured under standard conditions in the presence of H2O2 300µM. Cell viability was evaluated by MTT assay, and membrane lipid peroxidation was assessed by measuring malondialdeyde (MDA) using the TBARs assay. Total phenols were determined with Folin-Ciocalteu reagent. Results. In DPPH assay, the antioxidant activity was exibited by both inflorescences (1/IC50 range: 4.35-4.72) and leaves infusions (1/IC50 range: 7.18-7.87). Treatment with H2O2 reduced cell viability to 25.27±10.81% and increased MDA to 134.72±24.58% of control values (n=9-4). Infusions from both inflorescences and leaves (0.0004-1mg/ml) significantly counteracted the effect of H2O2 on both parameters. Leaves (49-49.36 mg/g DW) and inflorescences (31.39-32.86 mg/g DW) phenolic content were directly correlated with antioxidant activity (r2 >0.95).Conclusion. Antioxidant properties of Achillea collina, confirm the nutraceutical properties of this plant, particularly for leaves infusions prepared according to the traditional use.



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P-08FREE RADICAL SCAVENGING ACTIVITY AND EFFECTS ON THE OXIDATIVE METABOLISM OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE OF MENTHA PIPERITA ESSENTIAL OILS

R. Bombelli, A. Luini, E. Carcano, F. Marino, M.L., Colombo*, A. Conti#, M. Cosentino, S. Lecchini

Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Varese, I; *Department of Pharmaceutical Science and Technology, University of Turin, I; #Alpine Foundation for Life Sciences, Olivone, CH

Background and Aim - Peppermint is commonly used in folk medicine to treat loss of appetite, common cold, bronchitis, sinusitis, fever, vomiting, and dyspepsia. Circumstantial evidence suggests that peppermint exerts antimicrobial activities including antibacterial and antifungal effects. Peppermint is however also a rich source of polyphenolic compounds which possess strong antioxidant properties. The aims of this study were to evaluate the free radical scavenging capacity of Mentha piperita oils and their effects on the oxidative metabolism of human polymorphonuclear leukocytes (PMNs), which represent the first line of defense against fungal and bacterial infections.Methodsand Results - Mentha piperita oils were obtained from steam distillation of dried plants of varieties 541 RAC and Limbourg, cultured in Valtellina and in Val Formazza (Northern Italy) and harvested in june and september. PMNs were isolated from venous blood of healthy donors by density-gradient centrifugation and cultured in standard conditions. In the DPPH antioxidant assay none of the oils displayed any significant antioxidant activity up to dilutions 1:500. In the linoleic acid/TBARS assay all the oils displayed significant antioxidant activity at dilutions higher than 1:10.000. The viability of PMNs, as assessed by the MTT assay, was not significantly affected by any of the tested oils at dilutions of 1:100.000 or higher and for up to 24-h exposition. Not cytotoxic dilutions of the oils had no significant effects on spontaneous ROS production. ROS production from fMLP-stimulated PMN was significantly increased by Mentha piperita oil of var. 541 RAC harvested in june at dilutions of 1:100.000 (130.2±21.7% of fMLP alone, p<0.0002). On the contrary other oils had no effects on this parameter.Conclusion – The various oils have weak but significant free radical scavenging action. However only the Mentha piperita oils var. 541 harvested in june was able to affect the oxidative metabolism of human PMN. The ability of this oil to increase ROS production by PMN may indeed result in strengthened antimicrobial activity and antifungal properties of Mentha piperita. Nonetheless the ineffectiveness of the other oils suggests the need to select the variety and the harvesting time for an effective use of these remedies.



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P-09IMMUNOMODULATORY EFFECTS OF DIFFERENT MENTHA PIPERITAVARIETIES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

Elena Carcano, Marco Cosentino, Raffaella Bombelli, Alessandra Luini, Franca Marino, § Maria Laura Colombo, #Ario Conti, Sergio Lecchini

Department of Clinical Medicine, Section of Clinical and Experimental Pharmacology, University of Insubria, Varese, Italy, §Department of Pharmacology Science and Technology, University of Turin, Italy, and #Alpine Foundation for Life Sciences, Olivone, Switzerland

Mentha have a well-established role in folk medicine in the treatment of several disturbances and essential oils of Mentha are used also as anti-inflammatory remedies. This study investigated the effects of Mentha piperita var. 541 (Mentha541) and var. Laimbourg (Mentha Laimbourg), harvested in two different fields (Valtellina and Val Formazza) and two harvest times (june and september), on the proliferation and the production of Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) cytokines in human peripheral blood mononuclear cells (PBMCs). Essential oils of Mentha Laimbourg harvested either in september or in june (1:1000-1:500) significantly reduced cell proliferation. A similar inhibitory effect was exerted by both Mentha 541 harvested in Valtellina in september (1:1000-1:500) and Mentha 541 harvested in Val Formazza in june (1:10000-1:500). On the contrary, Mentha541 harvested in Valtellina in june had no effect at all. No effect was observed on the production of cytokines in the presence of any of the essential oils tested, however Mentha 541 harvested in both Valtellina and Val Formazza in june (1:10000-1:1000) significantly increased the ratio IFN-γ/IL-4 (P<0.01). The differences observed may be accounted for by differences in the composition of essential oils.



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P-10IN VITRO ANTI -TUMORAL ACTIVITY OF PISTACIA LENTISCUSPHYTOCOMPLEXES

Alessandro Buriani, Stefano Fortinguerra, Anna Signorelli, Gabbriella Innocenti1, Stefano Dall’Acqua1, Mariagnese Barbera2, Anna Caputo2, Maria Carrara2

Data Medica Padova Research Labs; 1Department of Pharmaceutical Science, 2Department of Pharmacology and Anaesthesiology, Padova University

Phytocomplexes, obtained from various specimens of Pistacia Lentiscus from the Mediterranean region, contain monoterpenes as their major components. A variety of monoterpenes have been shown to be effective in the chemo-prevention and chemotherapy of cancer, and they are going to be tested in clinical trials for their chemotherapeutic activity. In this study we investigated the biological activity of phytocomplexes obtained by hydrodistillation from Pistacia lentiscus (leaves, berries, flowers and branches) and Pistacia lentiscus var. chia(mastic gum) of various origin. We assessed the chemical composition of the phytocomplexes by gas-chromatography and mass spectroscopy, whereas their cytotoxic effect was evaluated by MTT reduction assay on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian) and LoVo (colon). Furthermore, we characterized the type of cell death in order to identify possible apoptotic mechanisms induced by phytocomplexes. The results indicate that, after 3 hours of treatment, the phytocomplexes were able to induce cytotoxic effects on all the three cell lines considered, and that the apoptotic mechanisms seems to be implicated in cell death. The only sample which did not induce any significant cytotoxic effect in our experimental conditions was the oil from Pistacia lentiscus var. chia. Although all phytocomplexes were rich in terpenic compounds, no consistent correlation between the composition and the cytotoxic activity of the samples was identified. Furthermore, no molecule singularly responsible for the activity of the phytocomplexes was detected, including terpens such as limonene, which is known to exert antitumor activity in different experimental models.



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P-11ANTIOXIDANT CAPACITY AND CHEMICAL CHARACTERIZATION OF HONEY FROM HERBS PRODUCED IN MOUNTAIN AREAS

A. Giorgi1, M. Madeo1, P. Francescato2, P. Cairoli2, G. Speranza2

1Dipartimento di Produzione Vegetale, Facoltà di Agraria – Via Celoria, 2 - Milano2Dipartimento di Chimica Organica e Industriale, Facoltà di Scienze M.F.N. - via Venezian, 21 – Milano. E-mail [email protected]

Introduction. Honey is a powerful sweetening agent and is used as a food preservative for its antioxidant properties. Its composition and quality depend on different parameters such as geographical and botanical origin, climatic and environmental conditions. A great number of medicinal plants are also used as a source of antioxidants that are associated with human health. Objective. The aim of this work was to evaluate the presence of secondary metabolites derived from Achillea millefolium in honey produced by bees placed in yarrow’s cultivations located in 3 different fields at above 1000 m a.s.l. in mountain areas. Methods. Antioxidant activity was estimated spectrophotometrically in the presence of the stable free radical DPPH. Phenolic compounds were extracted using Amberlite XAD-2 resin and a RP-HPLC method involving gradient elution and UV detection was applied to their separation. Total phenolic content was evaluated using Folin-Ciocalteau reagent. Results and conclusion. Results show that the radical scavenging capacity of honey is correlated to its colour intensity. All the honey samples present a characteristic chemical fingerprint that confirm usefulness of the phenolic profile to define their botanical origin. Furthermore a very good correlation was found between phenolic profiles of honeys from different fields in which the same botanical species were cultivated. This results suggest that honey could represent a real source of useful and healthy secondary metabolites when obtained from specific herbs, increasing its nutraceutical value.



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P-12ANTI-INFLAMMATORY EFFECTS OF A TRADITIONAL KOREAN HERBAL MEDICINE, SCUTELLARIAE RADIX

Inhye Ham1, Yunmi Ko1, Juyeong Lee1, Gabsik Yang1,Kyesup Jun2,Jonghoon Park2, Chanjung Park2 , Hoyoung Choi1

1 College of Oriental Medicine, Kyunghee University, Dongdaemun-gu, Seoul, 130-701, Korea 2 Woongjin Coway Co.,LDT., Euljiro 2-Ga, Joong-Gu, Seoul, 100-844, Korea

Dried root of Scutellaria baicalensis has been widely used as an anti-inflammatory agent in traditional Korean herbal medicine. Several studies indicated that extract and some compounds from Scutellariae radix have anti-inflammatory effect. However, the mechanism underlying anti-inflammatory activity was unclear. To evaluate the possible mechanisms responsible for the anti-inflammatory effect of crude extract and the lipophilic / hydrophilic fractions of Scutellariae radix, cytokines from lipopolysaccharide (LPS)-induced inflammatory response HepG2 cells, NO synthesis and cyclooxygenase (COX)-2 expression were measured. TNF-α, IL-1β and IL-6 were down regulated significantly when LPS-induced HepG2 cells were cultured in the presence of the lipophilic fraction of Scutellariae radix. The lipophilic fraction also showed considerable inhibition of NO production and COX-2 expression. This study indicated that Th1 cytokines, IFNγ and IL-12, were down regulated, while Th2 cytokines, IL-4 and IL-10, were up regulated upon treatment with crude extract and lipophilic / hydrophilic fraction of Scutellariae radix.

Acknowledgement : Woongjin Coway CO.,LTD.

References:1. B. Gayathri, N. Manjula, K.S. Vinaykumar, B.S. Lakshmi, A. Balakrishnan (2006), International Immunopharmacology 7: 473–4822. D.A. Israf, T.A. Khaizurin, A. Syahida, N.H. Lajis, S. Khozirah (2007), Molecular Immunology 44 : 673–679



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P-13HEALING EFFECTS OF ALCHORNEA TRIPLINERVIA ON ENHANCE THE COX2 AND PROSTAGLANDIN E2 EXPRESSION

Zeila P. Limaa, Tamara R. Calvob, Elisangela F. Silvac, Claudia H. Pellizzond, Wagner Vilegasb, Alba R.M.S. Britoc, Clélia A. Hiruma-Limaa

aDepartment of Physiology, Biosciences Institute, cp.510, São Paulo State Univesity, Botucatu, SP, CEP 18618-000, Brazil; bDepartment of Organic Chemistry, Chemical Institute, Universidade Estadual Paulista, Araraquara, SP, Brazil; cDepartment of Physiology, Biology Institute, Universidade Estadual de Campinas, Campinas, SP, Brazil; dDepartment of Morphology, Biology Institute, São Paulo State University SP, Brazil.

Alchornea triplinervia is a medicinal plant used in folk medicine as infusion to treat pain, ulcer, and gastric pain in general. We selected the lower effective dose of ethyl acetate fraction of A. triplinervia leaves (EAF-100 mg/kg), cimetidine (100 mg/kg) or saline as vehicle (10 ml/kg). EAF exerts gastroprotection by increase prostaglandins levels in rats. EAF was also effective in promoting the healing process in chronic gastric ulcer induced by acetic acid in rats. Three groups of male Wistar rats fasted for 24 h were used in this experiment (n=6-8). Under anaesthesia, a laparotomy was done in all animals through a midline epigastric incision. The acetic acid solution 0.05 mL (v/v) of 30% was injected into the subserosal layer in the glandular part of the stomach. The abdomen was then closed and all treatments were done orally once a day during 14 consecutive days beginning one day after surgery. After the sacrifice of animals the lesion was sectioned, and fixed in ALFAC solution (alcohol, chloroform and acetic acid) for 24 h in 4º C. Then the samples were routine processed for embedding in paraplast, and cut into 7µm thick section. These sections were stained with hematoxylin and eosin and immunohistochemistry were made using specifics mouse polyclonal antibody for COX2 cells, G, D cells and CXCR4. EAF presents a potent stimulator of gastric epithelial cell proliferation that contributed to acceleration of gastric ulcer healing induced by acetic acid. In imunohistochemical analysis we observed a large number of COX2 cells main in the submucosa and the results shows that G cells declined while D cells increased. We also studied the effects of treatments with EAF on angiogenesis during gastric ulcer healing, by CXC chemokine receptor 4 (CXCR4) expressions in the gastric mucosa. EAF increased the expression of CXCR4 and marked increase the formation of blood vessels supplying the stomach that increase the healing effect of gastric ulcer in rats. We established the phytochemical profile of EAF that contains flavonoids and these compounds may contribute to the observed antiulcerogenic effect of this specie.

Keywords: Alchornea triplinervia; antiulcer activity; prostaglandin E2; CXCR4; COX2.



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P-14DAVILLA NITIDA: ROLES OF ENDOGENOUS SULPHYDRYLS AND NO IN GASTRIC PROTECTION AGAINST LESIONS INDUCED BY ETHANOL

Helio Kushimaa, Clenilson M. Rodriguesb, Lucia R.M. Rochaa, Maira Colac, Alba R.M.S. Britoc, Daniel Rinaldob, Lourdes C. Santosb, Wagner Villegasb, Clélia A. Hiruma-Limaa

a Department of Physiology, Biosciences Institute, cp.510, São Paulo State Univesity, Botucatu, SP, CEP 18618-000, Brazil.b Department of Organic Chemistry, Chemical Institute, Universidade Estadual Paulista, Araraquara, SP, Brazil c Department of Physiology, Biology Institute, Universidade Estadual de Campinas, Campinas, SP, Brazil.

The medicinal plant study is a way to find news drugs to treat illness that still don’t have a good treatment or which the medicines used, provoke very hard side effects, incompatible to the benefits. Through quimiotaxonomics studies Davilla nitida (Dilleniaceae) was selected to evaluate potential antiulcerogenic effect and its mechanisms involved. The aim of this study was to verify the roles of endogenous oxide nitric (NO) and sulphydryls compounds on the gastric protective effect promoted by a single dose (500 mg/kg) of metanolic extract of D. nitida (EDN) in ethanol induced ulcer model in Wistar rats. This dose selected by previous studies of dose-response curve that indicated 500 mg/kg the best dose. To assess these aims were used two drugs NEM (a sulphydryl blocker) and L-NAME (a NO blocker) applied intraperitoneally before administration of EDN. After EDN oral administration, the gastric lesions were induced with I mL of ethanol orally. The experiment with L-NAME, EDN promoved a reduction of gastric lesions in 84% (p < 0.01), when compared to saline group. It is very similar result obtained with EDN without L-NAME application that was 90% of protection, compared to another group saline without the blocker. In the presence of NEM, EDN wasn’t able to prevent the gastric lesions (p > 0.05) and gastric protection was 31% compared to saline. In the group treated with EDN, without NEM administration, the extract was able to protect (p < 0.01) in 96%, when compared to another saline group without NEM. These experiments shown that EDN in the dose of 500 mg/kg is able to protect in ulcers induced by ethanol and this protection to cover the sulphydryl compounds.

Acknowledgements: FAPESP/BIOTA, CNPq.



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P-15GASTRIC ULCER HEALING PROMOTED BY FLAVONOIDS AND TANNINS FROM THE MEDICINAL PLANT MOURIRI PUSA GARDN.(MELASTOMATACEAE)

Paulo César de Paula Vasconcelosa; Márcio Andreob; Clélia Akiko Hiruma-Limac; Wagner Vilegasb; Cláudia Helena Pellizzona

aDepartment of Morphology, Biology Institute, São Paulo State University SP, Brazil; bDepartment of Organic Chemistry, Chemical Institute, Universidade Estadual Paulista, Araraquara, SP, Brazil; cDepartment of Physiology, Biology Institute, Universidade Estadual de Campinas, Campinas, SP, Brazil.

Mouriri pusa (MP), popularly known as “manapuçá” or “jaboticaba do mato”, is a plant from Brazilian cerrado that is commonly used in the treatment of gastrointestinal disturbs. This plant has shown intense gatroprotector effect. The present work was carried out to investigate the effect of tannins and flavonoids fractions from MP leaves methanolic extract in the cicatrisation process of gastric ulcers, and also evaluate possible toxic effects. For this, we used male Wistar rats (150-200g) . The gastric ulcers were induced by subserosal injection of acetic acid. The animals were divided in four groups: saline (S) 10ml/kg, cimetidine (CIM) 100mg/kg, tannins fraction (TF) 25 mg/kg and flavonoids fraction (FF) 50mg/kg. The rats received the treatments orally for 14 days. Body weight was recorded daily. After the treatments, the rats were killed. The stomachs were removed and the pH was measured, as well as the lesion (macroscopicaly). The rats’ blood was collected for toxicity analysis of AST, ALT, Gamma-GT, creatinine and urea. Internal organs were weighed. The injured part of the stomachs was sectioned and routine processed to be put on slides. These slides were used to hematoxylin and eosin staining, PAS staining and PCNA, Cox-2 and CXCR4 immunohistochemistry. The cicatrised part of the lesions was found significantly bigger in TF group by macroscopic measures. The height of regenerative mucosa was measured microscopically, revealing that it was significantly higher in FF (1337.1 ± 32.2µm) and TF (1397.9 ± 34.2µm) groups comparing to control (1055.4 ± 41.8µm). PCNA immunohistochemistry revealed greater number of dividing cells in FF group (870.30 ± 56.8) than in S group (327.46 ± 41.4 cells/mm²). CXCR4 immunohistochemistry revealed enhancement in angiogenesis by FF (78.45 ± 6.0 vessels/mm²) than by S (47.63 ± 4.1 vessels/mm²), while COX-2 was decreased by FF (2.99x10³ ± 519.0µm²/field) and FT (5.96x10³ ±

627.83µm²/field) than by S (13.89x10³ ± 1805.6µm²/field). These data denote that both fractions were effective to enhance the gastric ulcers healing. Moreover, there was no toxic effect observed concerning biochemical analysis, body weighing and internal organs weighing.

Acknowledgements: BIOTA/FAPESP and CNPq



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P-16FENNEL TEA IN INFANCY – A BENEFIT-RISK-EVALUATION

Felix Iten, Reinhard Saller

Inst. für Naturheilkunde, Universitätsspital Zürich

In the Chinese and the European culture fennel seeds have a long lasting tradition as a spice and as a remedy. Even today, fennel tea belongs to the most frequently used remedies against gastrointestinal complaints especially in infants and children. Its popularity is primarily based on its effectiveness and its tolerability. Therefore, the note of the German BgVV from May 11th 2001, recommending the consumers to limit the consumption of fennel tea because of an assumed carcinogenic risk, unsettled many consumers. The warning was exclusively based on animal experiments that suggested a cancer risk due to estragole a component of the essential oil of fennel seeds. However, direct translation of animal experiments to the human situation is problematic for numerous reasons. In addition, no clinical, epidemiological or experimental evidence exists, that would confirm the supposed cancer risk. A critical evaluation of the respective studies leads to the conclusion, that a cancer risk, should it really exist, would be negligible. For the majority of the beneficial aspects, traditionally attributed to fennel seeds, clinical and experimental evidence are available by now. Therefore, we are able to endorse fennel tea as safe and to recommend the consumer to go on drinking fennel tea.



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P-17PRECLINICAL EVALUATION OF STANDARDIZED EXTRACT OF MUCUNA PRURIENS IN ANIMAL MODELS OF PARKINSON’S DISEASE

Kasture Sanjay1, Pontis Silvia2, Simola Nicola2, Pinna Analisa2, Liliana Spina2, Rosanna Longoni2, Morelli Micaela2

1* - MGV’s Pharmacy College, Panchavati, Nashik, INDIA, 2 - Department of Toxicology, University of Cagliari, via Ospedale, Cagliari, ITALY

Mucuna pruriens is used in the Ayurvedic medicine to treat Parkinson’s disease (PD). The standardized extract of Mucuna pruriens contained 12.5 % levodopa. In the present study, we compared the antiparkinson’s activity of standardized extract of Mucuna pruriens with levodopa, using animal models of motor impairment such as rotational behavior in 6-hydroxy dopamine lesioned rats and stepping test, or models of PD tremor as tacrine-induced tremulous jaw movements. The levodopa decarboxylase inhibitor benserazide (6 mg/kg) was administered intraperitoneally (i.p.) 30 min before levodopa or the extract. Similarly to levodopa (6 mg/kg i.p.), the Mucuna pruriens extract (48 mg/kg i.p., a dose equivalent to 6 mg/kg of levodopa) induces the onset of contralateral turning behavior in 6-hydroxydopamine lesioned rats, the effect persisting for longer time as compared to levodopa. The extract showed a weak rotational behavior in absence of benserazide, whereas, levodopa was ineffective in absence of the decarboxylase inhibitor. Subchronic administration of the extract exhibited abnormal involuntary movements, not statistically different from those produced by an equivalent dose of levodopa. Moreover, the extract reduced tacrine-induced jaw movements, improved the performance in initiation of stepping and increased the number of adjusting steps. The extract did not exhibit place preference in a dose equivalent to 20 mg/kg of levodopa. In conclusion, the study justifies use of Mucuna pruriens in the treatment of Parkinson’s disease.



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P-18ANTIOXIDANT EFFECT OF THE ETHYL ACETATE EXTRACT OF TEUCRIUM POLIUM L. ON CCL4 INDUCED DAMAGE IN RAT’S LIVER

Tatjana Kadifkova Panovska1, Svetlana Kulevanova2, Icko Gjorgoski3, Mirjana Bogdanova4 and Gordana Petrushevska5

1Institute of Toxicology, Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Skopje, Macedonia2Institute of Pharmacognosy, Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Skopje, Macedonia3Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, Skopje, Macedonia4Institute of Clinical Biochemistry, Clinical Center, Skopje, Macedonia5Institute of Pathology, Faculty of Medicine, University “Ss. Cyril and Methodius”, Skopje, Macedonia

The antioxidant activity of ethyl acetate extract of Teucrium polium (L.) was investigated using rats with CCl4-induced liver damage. Specific biochemical parameters (glutathione peroxidase, superoxide dismutase, reduced glutathione and total antioxidative status) were estimated in blood and in liver homogenate. Lipid peroxidation in CCl4-intoxicated rats was evidenced by a marked increment in the levels of TBARS. Histopatological examinations of the liver were undertaken to monitor the status of the liver. Silymarin was used as a standard to compare the hepatoprotective activity of the extract. The biochemical parameters in groups treated with Teucrium polium extract at a dose of 25 mg kg-1, have shown significantly different value than that of the CCl4-treated group. The liver biopsy of all experimental rat groups treated with Teucrium polium ethyl acetate extract showed significant restoration of normal histomorfological pattern of liver cells. The study revealed that T. polium extract have a potential to prevent oxidative damage induced by CCl4 in liver.



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P-19A NEW MODEL FOR STUDY OF HYPO- AND HYPERTENSIVE EFFECTS IN OVO

Makarova M., Selesneva A., Makarov V.

St-Petersburg Institute of Pharmacy, 27-424, Partizanskaya str, 195248, St.-Petersburg, Russia

Application of the modern methods allows visualisation of reaction of vessels on irritation and measuring of its diameter in real time. The HET-CAM (Hen Egg Chorioallantoic Membrane) test was proposed for testing of irritating effect of hypotensive and hypertensive drugs in the chorioallantoic membrane of a chicken.Eggs of White Leghorn chickens were used in the age of 9-10 days. Eggs shells were opened accurately. It was observed that diameter of CAM vessels of I, II, III and IV orders remain unchanged during 180 minutes. Ordinary vasaconstrictor - norepinephrin (0.1 µM) was dipped on CAM. The most constriction was observed in first 5 minutes. The diameter of vessels of I and II orders decreased in 31 % to the control. Diameter of vessels III and IV orders decreased in 57.2% and 60.0 % respectively.In other experiment hypotensive drug atenolol (0.4 mM) was applied on CAM. Norepinephrin was dipped in system after 2 minutes. Atenolol have prevented the development of vasaconstrictor effect of norepinephrin. And diameter of all vessels not changed in 60 minutes. Thus, the test for the CAM can be applied in biological researches to studying hypo-and hypertensive effects of drugs.



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P-20AN UPDATED SYSTEMATIC REVIEW WITH META-ANALYSIS OF THE CLINICAL EVIDENCE OF SILYMARIN

R. Saller MD1, R. Brignoli MD2, J. Melzer MD1, R. Meier MD3

1 Institute of Complementary Medicine, Department of Internal Medicine, University Hospital Zurich,2 Clinical pharmacology - Tradyser GmbH, Rueschlikon ZH,3 Department of Gastroenterology, University of Basel, Liestal, Switzerland

Background: The potential benefit of silymarin (special extract from the fruits of Silybum marianum) in the treatment of liver diseases remains a controversial issue.Methods: For this systematic review electronic databases identified 64 papers for the search terms silymarin, silibinin, silicristin or milk thistle and clinical trial. Only 18 complied with the criteria ‘double-‘ or ‘single-blind’. These publications were analysed from a clinical point of view and meta-analytic calculations were performed.Results: The clinical evidence of a therapeutic effect of silymarin in toxic liver diseases (except the efficacy in Amanita phalloides poisoning) is scarce. There is no evidence of a favourable influence on the evolution of viral hepatitis, particularly hepatitis C. In alcoholic liver disease, comparing with placebo, aspartate aminotransferase was reduced in the silymarin treated groups (p = 0.01) while alkaline phosphatase was not. In liver cirrhosis, mostly alcoholic, total mortality was 16.1 % with silymarin, vs. 20.5 % with placebo (not significant); liver related mortality was 10.0 % with silymarin, vs. 17.3 % with placebo (p = 0.01). In patients with diabetes and chronic alcoholic liver disease, the drug caused a significant decrease in both glucose and triglyceride plasma levels. Conclusions: Based on the available clinical evidence it can be concluded -concerning possible risks / probable benefits – that it is reasonable to employ silymarin as a supportive element in the therapy of mushroom poisoning but also (alcoholic and grade Child ‘A’) liver cirrhosis. A consistent development program, consolidating existing evidence and exploring new potential uses, would be very welcomed.



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P-21INFLUENCE OF ISOFLAVONES ON THE PROTEIN EXPRESSION IN HUMAN LIVER (Hep G2) AND BREAST (MCF 7) CANCER CELLS – A PROTEOMIC APPROACH

G. Pakalapati, M. Wink

Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.

IntroductionRecently, isoflavones have received profound attention for their potential health benefits. The intake of soya isoflavones (genistein, daidzein and glycitein) and red clover isoflavones (genistein, daidzein, formononetin and biochannin A) associate with the biological effects. It is known that isoflavones can bind to estrogen receptors and modulate the activity of estrogen regulated genes. Some are strong inhibitors of tryrosine kinases and can induce apoptosis.In our study the toxic activities and mechanisms underlying the molecular and cellular effects in commercially available products from soya (Glycine max) and red clover (Trifolium pratense) were tested in human liver (HepG2) and breast cancer cell lines (MCF7).MethodsCytotoxicity of soya and red clover extracts were assessed using MTT metabolic assay. Cells were incubated for 48 hours with 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml of extracts. Scavenging activity of stable DPPH free radicals was done to evaluate the antioxidant activity of the extracts. Proteomic study (2D gels) was performed on cells, treated with effective concentration (EC50) to observe the protein regulation and expression profile. Gels were stained with coomassie blue and silver stain and differentially regulated proteins were determined by mass spectrometry (MALDI-TOF-MS).ResultsSoya extract did not show any cytotoxicity even at high concentration contrasting to red clover which showed significant toxicity. A considerable DPPH radical scavenging activity was also shown by red clover than soya. 2D gels showed a differential protein profile between treated and untreated cells.ConclusionsThe study showed detectable moderate modulations in the proteome of treated and untreated cells. Proteomics can be a promising field to understand and evaluate the biological activities of soya and red clover, although it can only detect changes of prominent proteins.



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P-22PHYTOECDYSTEROID COMPOSITION OF MICROSORUM SPECIES (POLYPODIACEAE) OF FRENCH POLYNESIA

R. Hoa, T. Teaia, D. Loqueta, J.-P. Bianchinia, J.-P. Giraultb, R. Lafontc, P. Raharivelomananaa

aLaboratoire de Chimie des Substances Naturelles, Université de la Polynésie Française, B.P. 6570 Faaa, 98702 Faaa, Tahiti, French Polynesia.bLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601, Université Paris 5 - René Descartes, 45 rue des Saints Pères, 75270 Paris, Cedex 06, France.cLaboratoire de Biochimie structurale et fonctionnelle des Protéines, CNRS FRE 2852, Université Paris 6 – Pierre et Marie Curie, Case 29, 7 Quai Saint-Bernard, 75252 Paris, Cedex 05, France.

Ferns have always played an important part in the everyday life of Polynesians as ornamental and medicinal plants. Among them, ferns belonging to the genus Microsorum (Polypodiaceae), e.g. M. scolopendria, are used in a great number of remedies in traditional medecine and contain phytoecdysteroids. Ecdysteroids are known to have many pharmacological (e.g. anabolic, hypoglycemic, and tonic) effects in humans.Six species of Microsorum are found in French Polynesia: M. scolopendria, M. membranifolium, M. maximum, M. punctatum, M. commutatum and M. rubidum. A chemical investigation of these species has been performed to establish their ecdysteroid content and pattern.Samples of fronds (and rhizomes) from the six species were collected and analyzed by HPLC for their ecdysteroid content. The main constituents were isolated and identified by spectroscopic techniques.Seven ecdysteroids were quantified in these species, among which the major ones are: ecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Interestingly, the highest ecdysteroid concentrations were found in the two species M. membranifolium and M. scolopendria, i.e. the ones mostly used in Polynesian traditional medicine. Both fronds of M. membranifolium and rhizomes of M. scolopendria grown in French Polynesia can indeed be considered as rich sources of ecdysteroids, especially of ecdysone, which is rarely present as a major component in plants.These findings support our hypothesis that the medicinal properties of the two species M. membranifolium and M. scolopendria, are at least in part connected with their high ecdysteroid content.



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P-23MEDICINAL PLANTS FROM FRENCH POLYNESIA: EVALUATION OF THEIR FREE RADICAL SCAVENGING ACTIVITY

T. Leua, S. Souleta, D. Loqueta, L. Meijerb, P. Raharivelomananaa

aLaboratoire de Chimie des Substances Naturelles, Université de la Polynésie Française, B.P. 6570 Faaa, 98702 Faaa, Tahiti, French PolynesiabStation Biologique de Roscoff (CNRS) Place Georges TESSIER –BP 74, 29682 Roscoff CEDEX France

French Polynesia, one well-known « hot-spot » world biodiversity, is an Overseas French territory having an original flora within more than 500 species used in traditional medicine. Plants have great heritage value in French Polynesia and many are still used in traditional medicine or for ritual ceremonies. Little is known about this biodiversity asset mainly in phytochemical and bioactivity standpoints.In order to contribute to the knowledge of biodiversity of French Polynesia, medicinal plant species were screened for the first time to determine their free radical scavenging and antioxidant activities. Free radicals are implicated in a number of diseases states that can have serious effect on the cardiovascular system and degenerative disorders (cancer, Alzheimer’s disease…).After an inventory of the most used medicinal plants, antioxidant activity screening was carried out on 33 species belonging to 24 families.Leaves and wood parts of the studied species were collected in Tahiti and extracted. The extracts were submitted to antioxidant test using DPPH assay.16 crude extracts exhibited scavenging properties toward the DPPH radical. These active extracts were fractioned following polarity partition. The antioxidant activity of the obtained fractions were quantified. Medium polarity fractions showed the best results. The most active plants were Weinmania parviflora, Macaranga taitensis and Metrosideros collina.The most active extracts will be subjected to further phytochemical studies to identify active components responsible of the shown bioactivities. Other specific activities will be assessed to these leads.



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P-24FUCAN SULFATE DIRECTLY INHIBIT THE ACTIVITY OF SERIN PROTEINASES

Cinzia Repice, Barbara Graziadio, Maria Distaso, Massimo Iacobelli, Cinara Echart

Gentium SpA., Villa Guardia (Co), Italy

Background: fucan sulfate are sulfated fucosylated polymers from brown algae cell wall that exhibit some heparin/heparan sulfate properties. They are among the most widely studied of all the sulphated polysaccharides of non–mammalian origin that exhibit biological activities in mammalian systems. Previously, we have shown that sulfated fucans with low molecular weight (14000-29000 Da) have anticoagulant and antithrombotic activities. Cathepsin G and Elastase are two serine proteinases contained in azurophil granules of polymorphonuclear leukocytes involved in thrombotic events. Cathepsin G act as a potent agonist of human platelet activation leading to their aggregation. While, Elastase degrades collagen, elastin and proteoglycans. Objectives: the scope of this study was to evaluate the effects of fucan sulfate on the catalytic activity of Cathepsin G and Elastase, in vitro.Methods: the catalytic activity of Cathepsin G and Elastase was measured in presence or absence of a wide range of concentration of fucan sulfate by continuous monitoring the release of ρ- nitroanilide from the specific chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA and MeOSuc-Ala-Ala-Pro-Val-pNA, respectively. Results: we demonstrate that fucan sulfate inhibit the activity of Cathepsin G and Elastase to degrade its specific substrate in a dose dependent manner.Conclusion: the present study demonstrates that fucan sulphate is able to inhibit the activity of Cathepsin G and Elastase in the hydrolysis of the chromogenic substrates. These findings suggest the antithrombotic activity of fucan sulfate.



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P-25HET-CAM ASSAY FOR VASODILATATION EFFECT OF NANOSYSTEM WITH TAXIFOLIN

1Pozharitskaya O., 1Karlina M., 1Shikov A., 1Makarova M.N., 2Tikhonov V.P.

1St-Petersburg Institute of Pharmacy, 27, Partizanskaya str, 195248, St.-Petersburg, Russia, 2 "Diod" Ltd, 11a Derbenevskaya ave., 113114, Moscow, Russia

The HET-CAM test system allows the simulation of capillary protective effects prior to actual animal testing.Taxifolin – flavonoid has a wide spectrum of biological activity: capillary protective, antioxidative, anti-inflammatory and antithrombotic [1,2]. Nanosystem in form of self-microemulsifying drug delivery system (SMEDDS) is perspective for oral, nasal and transdermal applications. The aim of the present work was to study vasodilatation effect of SMEDDS with taxifolin in ovo (Het-Cam assay). A nanoemulsion containing 2% taxifolin, surfactant, cosurfactant, oil phase and water was prepared. Eggs of White Leghorn chickens were used in the age of 9-10 days. On 0.3 ml of tested compositions were applied on chorioallantoic environment of chicken embryo. Vasodilatation effect was evaluated on change of diameter of vessels in 60 s using the HET-CAM assay. It was shown, that SMEDDS of taxifolin has significant vasodilatation effect. Its application increases diameter of intact vessels of the second order by 9 %, the third order by 14 % and the fourth order by 19 %. The data have allowed assuming, that vasodilatation effect of SMEDDS is associated not only with taxifolin, but also with oil phase.

References: 1. Middleton E. et al. (2000) Pharmacol. Rev. 52: 673-751. 2. Silva M.M. et al (2002) Free Radical Res. 36 (11):1219-1227.



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P-26ANTIULCER AND HEALING PROPERTIES FROM TWO VERNONIA POLYANTHES LESS. FRACTIONS

Victor Barbastefanoa; Maíra Cola-Mirandaa; Anderson Luiz-Ferreiraa; Elizangela Farias-Silvaa; Christiane Takayamaa; Daniel Rinaldob; Clélia A. Hiruma-Limac; Wagner Vilegasb; Alba R. M. Souza Britoa

a Departamento de Fisiologia e Biofísica, IB, UNICAMP, Campinas, SP, Brazil. b Departamento de Química Orgânica, IQ, UNESP, Araraquara, SP, Brazil.c Departamento de Fisiologia, IB, UNESP, Botucatu, SP, Brazil.

Background: The chloroformic crude extract from Vernonia polyanthes Less. (Asteraceae), a Brazilian savannah plant popularly known as “assa-peixe”, had been already investigated for its antiulcerogenic properties. The outstanding results obtained leaded our lab to evaluate two organic fractions from this chloroformic crude extract. Objectives: The methanolic and chloroformic fractions (MVP and CVP) from the chloroformic crude extract of Vernonia polyanthes were investigated for its antiulcerogenic and healing properties employing two experimental models. Methods: To obtain MVP and CVP, the chloroformic crude extract from Vernonia polyanthes was fractionated by column chromatography (Silica Gel 60). The antiulcerogenic activity of MVP and CVP, in the doses of 3, 6 and 12 mg/kg, was evaluated in male Wistar rats, with the absolute ethanol-induced gastric ulcer model (1 mL, 1 hour before sacrifice). The healing properties and in vivo toxicity (weight of animals and its organs) of the best active fraction was achieved with the acetic acid (30%, 50µL)-induced ulcer model, where rats are sacrificed just after a 14 days treatment period. Results: Just MVP presented significant antiulcerogenic activity in lower doses (3, 6 and 12 mg/kg). The lowest dose (3 mg/kg), which presented 77,6% of ulcer inhibition, were then evaluated for its healing activity. MVP treatment for 14 days reduced in 75,2% the ulcer area in acetic acid-induced gastric ulcer and showed no apparent in vivo toxicity. Conclusions: MVP protects the gastric mucosa in a very low dose, in both acute and sub-acute experimental models, with no apparent signals of in vivo toxicity.



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P-27EFFECT OF THE INDIGO FROM INDIGOFERA TRUXILLENSIS ON COX-1 EXPRESSION IN THE GASTRIC MUCOSA OF RATS

Elisangela Farias-Silvaa; Tamara Regina Calvob; Débora de Paula Michelattoa, Ana Cristina Alves de Almeidaa, André Schwambach Vieiraa; Victor Barbastefanoa; Maíra Cola-Mirandaa; Anderson Luiz-Ferreiraa; Hiruma-Limac; Wagner Vilegasb; Alba R.M. Souza Britoa

a Departamento de Fisiologia e Biofísica, IB, UNICAMP, Campinas, SP, Brazil. b Departamento de Química Orgânica, IQ, UNESP, Araraquara, SP, Brazil.c Departamento de Fisiologia, IB, UNESP, Botucatu, SP, Brazil.

Background: According to previous data obtained in our laboratory, Indigofera truxillensis Kunth (Fabaceae), a Brazilian savannah plant popularly known as “anieira”, showed antiulcerogenic activity, involving increase on the PGE2 levels and mucus production. COX-1 appears to be responsible for the production of prostaglandins (PG) that are important for homeostatic functions, such as maintaining the integrity of the gastric mucosal. Indigo alkaloid, isolated from I. truxillensis, also presented antiulcerogenic activity, but its gastroprotective action mechanisms have not been investigated yet. Objective: Evaluate the influence of the Indigo alkaloid on the COX-1 expression in gastric mucosal from rats submitted to ethanol-induced gastric lesions. Methods: Male Wistar rats were pre-treated with Indigo (2 mg/kg), saline (vehicle) or rutin (200mg/kg, positive control) and submitted to absolute ethanol-induced gastric ulcer model (1 mL, 1 hour before sacrifice). Gastric mucosal was removed and homogenized with appropriated buffer and Western blotting procedures were employed to detect COX-1 expression. Results: Indigo (2 mg/kg) significantly up-regulates the COX-1 expression in gastric mucosal from rats. Conclusions: The Indigo alkaloid, isolated from Indigofera truxillensis, is an important compound e involved on the gastroprotective effects of the Indigofera truxillensis.



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P-28BIOASSAY-GUIDED FRACTIONATION OF THAI MEDICINAL PLANTS USED TO TREAT CANCER

I. Techatanawat(1,2), P.J. Houghton(2), P.J. Hylands(2)

1Government Pharmaceutical Organisation, 75/1 Rama VI Road, Ratchatewi, Bangkok 10400, Thailand2Pharmacognosy Research Laboratories, Pharmaceutical Sciences Research Division, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom

Eight Thai medicinal plants traditionally used to treat cancer were selected and extracted, according to traditional methods, to obtain water and alcoholic extracts.The extracts were tested in vitro for cytotoxicity using the Sulphorhodamine B (SRB) assay to assess cell growth inhibition (1) against normal cells (SVK-14, human keratinocyte) and 4 cancer cell lines (COR-L23, large cell lung carcinoma; MCF7, human Caucasian breast adenocarcinoma; C32, human amelanotic melanoma and CACO-2, human colon carcinoma). Of the 8 Thai medicinal plants, an ethanol extract of Ammannia baccifera (Lythraceae) showed the strongest cytotoxicity against both cancer cell lines and normal cells. It exhibited the lowest IC50 against COR-L23 (27.12 ± 1.40 µgmL-1 for exposure period and 19.95 ± 0.87µgmL-1 for recovery period). It was subjected to column chromatography over silica gel eluted with n-hexane: dichloromethane: methanol (step gradient) and multi-preparative thin-layer chromatography. (-)-(4R)-Hydroxy-1-tetralone, (-)-(4S)-acetoxy-1-tetralone, (-)-(4S)-hydroxy-1-tetralone-4-O-β-D-glucoside, β-sitosterol and β-sitosterol-β-D-glucoside have been purified and identified by the joint application of UV spectroscopy, mass spectrometry, NMR spectroscopy, specific rotation and circular dichroism spectroscopy. Further light on the precise nature of the shape of the molecule was provided by a more detailed analysis of the 1H NMR spectrum. Since the cyclohexanone ring was flexible, the conformation of the substituted group at C-4 of 3 tetralone derivatives was an average of the pseudoaxial and pseudoequatorial orientation (2). The absolute configuration and conformation responsible for the NMR measurement of (-)-(4R)-hydroxy-1-tetralone, (-)-(4S)-acetoxy-1-tetralone and (-)-(4S)-hydroxy-1-tetralone-4-O-β-D-glucoside were β-equatorial, α-axial and α-axial orientation respectively.

Acknowledgements: I. Techatanawat thanks the Thai Government Pharmaceutical Organisation for financial support

References: 1. Lin, Z. X. et al (1999) J. Ethnopharmacol. 66(2):141-50; 2.Talapatra, S. K. et al (1988) Phytochemistry 27: 3929-3932



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P-29INHIBITORS OF ACETYLCHOLINESTERASE FROM GENTIANA AND PEUCEDANUM SPECIES

Aurélie Urbain, Andrew Marston, Kurt Hostettmann

Laboratory of Pharmacognosy and Phytochemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland

Alzheimer’s disease (AD) is the most common form of dementia in elderly people. In patients with AD, the enzyme acetylcholinesterase (AChE) decreases neurotransmission by degrading acetylcholine, and furthermore accelerates brain lesions. Even though AD has no cure today, the use of approved AChE inhibitors allows cognitive improvement. A number of plant constituents, most notably alkaloids, have been found to inhibit AChE activity, and galantamine from Galanthus nivalis (Amaryllidaceae) is a drug of choice for the treatment of AD symptoms. However current approved medication still presents some disadvantages such as short half-lives or side effects.A screening program of plant extracts was undertaken in an effort to discover new, non-alkaloid inhibitors of AChE, on the assumption that compounds other than alkaloids could be as efficient while being safer.The extracts were screened using a TLC bioautographic assay. The bioactive compounds were then isolated using almost exclusively centrifugal partition chromatography. Structure elucidation was performed by a combination of mass spectrometry and 1D- and 2D-NMR.Three plant extracts exhibited significant inhibition of AChE activity: two Gentianaceae, Gentianella campestris (L.) Börner and Gentianella amarella subsp. acuta J.M.Gillett, and masterwort, Peucedanum ostruthium (L.) Koch (Apiaceae). From gentians, 9 xanthones with AChE inhibitory activity were isolated, whereas 5 active coumarins plus a chromone were obtained from masterwort. Thus, a total of 15 compounds exhibiting AChE inhibitory activity, belonging to 3 different classes of secondary metabolites, have been isolated from common Alpine plants. Therefore, these plants could be considered as potential sources for the treatment of neurodegenerative diseases.



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P-30ANTIDEPRESSIVE EFFECTS OF IGNATIA AMARA CONTAINING COMPLEX HOMEOPATHIC REMEDIES

Wasilewski B.W.

Department of Psychosomatics, Sexology and Pathology of Interpersonal Relations, Medical Centre for Postgraduate Education, Warsaw, Poland. e-mail: [email protected]

Objective: The purpose the successive work was evaluation of the therapeutic effects and side-effects in the use of the Ignatia amara containing preparation (Ignatia Homaccord) in the treatment of acute depression in women during the menopausal period and comparison of results obtained with that in the control group subjected to treatment with Fluvoxamine. Research tools and methodology: to measure the intensification of depressive disorders during the investigations, a 17 point version of Hamilton’s Depression Scale (HDRS) and Beck’s Self-report depression scale (BSRDS) were used. Results: The results of the comparative investigation of Fluvoxamine and Ignatia Homaccord, which covered 211 patients with depression ( ICD: F-32; F-33 ) connected with menopausal symptoms, showed the effectiveness of both preparations is similar, while the frequency of side effects during treatment with the Ignatia Homaccord preparation is significantly smaller and corresponds to the frequency observed with the administration of a placebo. A significantly smaller number of cases of interrupted treatment and much better drug tolerance were noted in the group of patients treated with Ignatia Homaccord. The results obtained induced the author to undertake pharmacological investigations concerning the analysed pharmaceuticals. Specific effects in pharmacological tests (open field test andelevated “plus” maze test ) confirmed psychotropic activity of Ignatia amara. Conclusions: Main benefit arising from the research conducted for this work is the identification of a new remedy with antidepressant activity, previously unidentified in the psychopharmacological literature.



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P-31LIPID LOWERING ACTIVITY OF CITRI UNSHII PERICARPIUM IN HYPERLIPEMIC RATS

Gabsik Yang, Juyeong Lee, Eui-Dong Jung, Yummy Ko, Byung-Hee Lee, Inhye Ham, Ho-Young Choi

* College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul, 130-701 Republic of Korea

Efforts to develop effective and better hyperlipemic drugs had led to the discovery of natural agents. In this study we sought to verify the usefulness of the hyperlipemia rat models induced by high cholesterol (HC)-diet, using Simvastatin (Simva), a well-known cholesterol lowering drug. Secondly, the cholesterol lowering effect (reduction in both plasma TG and TC) of Citri unshii pericarpium(CU) was evaluated using the same models. The inhibitory effect of the MeOH extract of CU and its fractions were tested in hyperlipemic rats using for animal models induced by high cholesterol-diet. We measured plasma levels of triglyceride, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol as measures of its hyperlipemic effects. We demonstrated that CU decreases plasma levels of triglyceride, total cholesterol and LDL cholesterol. There was also no elevation of plasma ALT and AST levels, which indicate CU did not cause liver injury. These results indicate that CU is a good candidate for the treatment on high cholesterol diet-induced blood circulatory disorders, obesity and hyperlipemia.

Key Words: Citri unshii pericarpium; High-cholesterol diet; Rat; Hyperlipemia; Body weight; Triglyceride; Low-density lipoprotein cholesterol; High-density lipoprotein cholesterol

Acknowledgements: This work was supported by the Second Stage of Brain Korea 21 project in Oriental Medical Science Center.

References:1. Ble-Castillo JL et al. Life Sci. 2002;70:2665.2. Wojcicki J et al. A search for a model of experimental atherosclerosis: comparative studies in rabbits, guinea pigs and rats. Pol J Pharmacol Pharm 1985; 37(1):11-21.3. Sacks FM et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators. N Engl J Med 1996;335(14):1001-9.



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P-32CHARACTERISATION AND ANTIOXIDANT EFFECTS OF ESSENTIAL OILS OF MENTHA CITRATA AND MENTHA PIPERITA

Zanni M., Bombelli R., Marino F., Cosentino M., Conti A.



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P-33NONSTEROIDAL ANTI-INFLAMMATORY PROPERTIES OF SOME ALPINE MEDICINAL PLANTS

Nadia Marcon, Stéphanie Berthouzoz, Alain F. Grogg, Frans Zonnevijlle

Institute of Life Technologies, University of Applied Sciences Valais, Rawyl 47, CH-1950 Sion

Background The determination in liquid medium of the in vitro nonsteroidal anti-inflammatory activity of all kinds of matrices has been the subject of quite a number of studies. Such measurements avoid but do not replace, in vivoexperiments on animals. Nevertheless they are very useful in systematic studies. They enable the quantification of the inhibition of certain enzymes that are responsible for the biochemical signal of inflammations, e.g. phospholipase A2, 5-and 12-lipoxygenase and cyclooxygenase.Commercially available standard microcuvette colorimetric tests work well with pure dissolved substances. However, the usefulness of these tests is limited when the active substance is present in a complex matrix, such as raw and refined plant extracts.Recently it has been shown for acetylcholinesterase [A. Marston, J. Kissling & K. Hostettmann: “A rapid TLC bioautographic method for the detection of acetylcholinesterase and butyrylcholinesterase inhibitors in plants“, Phytochemical Analysis 13 (1), 2002, 51], that it is possible to apply an enzymatic reaction to a thin layer, after separation of the matrix, in order to characterize by colorimetry the inhibitory effect of each individual fraction. Objectives As the characterization of the nonsteroidal anti-inflammatory activity without previous separation of a plant extract is questionable, it is proposed to transfer commercial microcuvette tests to thin layers in order to be able to compare directly raw plant extracts when carrying out a series of comparative measures.Methods Applying such enzymatic tests requires partial separation of theconstituents by TLC, and colorimetric detection in situ by spraying well-defined quantities per surface unit of a solution of the enzyme, the substrate (lipoxygenase inhibition) or the developer (phospholipase A2). A second treatment may be needed after fixation of the reagents (cyclooxygenase). Results The global in vitro nonsteroidal anti-inflammatory activity is presented, based on the IC50 values obtained for each specific enzymatic reaction. The results obtained for microcuvettes and for thin layers will be compared. Preliminary comparative results obtained for screening tests of raw extracts of a dozen plants will be presented. Conclusions The results obtained so far confirm the advantages of the proposed method.



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P-34POPULAR USES AND KNOWLEDGE ABOUT MEDICINAL PLANTS IN THE TICINO REGION

Giulia Poretti Lozano Becerra, Ario Conti

Alpine Foundation for Life Sciences, 6718 Olivone (Switzerland), Université de Neuchâtel, Faculté des Sciences (Switzerland)

An ethnopharmacobotany survey (PhD) is carried out in Canton Ticino (southern Switzerland region), with an approximated area of 2'812 km2 and a population of 322'700. The survey concerns two aspects of popular uses of medicinal plants : the oral traditional knowledge collection through interviews and the written material scrutiny. The written sources belong mainly to the papery archive of the dialetology and ethnographical Centre of Italian Switzerland (Centro di dialettologia ed etnografia della Svizzera italiana). The union of oral and written information aims to draw a repertory of popular medicinal plants in Ticino and a geographical panorama of their traditional therapeutical uses in human as well as in animal care. Data are collected in ethnobotanical papers, organized in alphabetical orders according to scientific specific names: every paper presents all the oral and written material concerning the medicinal plant. Tables resume and synthesize the therapeutical uses and the ethnophytonymes according to the 8 regional districts. For oral sources, the number of informants and the frequency of citation are also reported. So far, data obtained concern 98 plant species belonging to 51 botanical families used for common health care through interviews with 85 informants. Main medicinal plants (for number of informants and number of citations) are Malva sp. (Malva sylvestris L.; Malva neglecta Wallr.) and Matricaria recutita L.. Main botanical families for number of species quoted are Lamiaceae, Rosaceae and Asteraceae. Autochthonous and sub-spontaneous species represent the main portion of medicinal plants quoted (89%); 6% is the percentage of quoted medicinal plants which only grow in culture, while 5% concern plants which don’t grow on the territory. 27.6% of the plants quoted were also used for veterinary purposes. Keywords: Ethnobotany ; Medicinal Plants ; Canton Ticino ; Vernacular uses and knowledge; Oral and written sources.



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Acquas E. 51Adhvaryu M.R. 52Adinolfi B. 53Aftab K. 54Alves de Almeida A.C. 77Andreo M. 65Armocida A. 14Arredondo F. 39Baege A.C. 25Bagetta G. 36Balduzzi M. 32Barbastefano V. 76, 77Barbera M. 60Baroffio C. 15Bartsch H. 23Bauer R. 28Becker H. 23Begum S. 54Benvenuti S. 53Berthouzoz S. 83Bianchini J.-P. 72Blasina F. 39Bogdanova M. 68Bombelli R. 30, 55, 56,

57, 58, 59, 82Brignoli R. 70Brito A.R.M.S. 63, 64Buriani A. 60Cairoli P. 61Calvo T.R. 63, 77Caputo A. 60Carcano E. 30, 56, 57,

58, 59Carlen C. 15Carnovale-Scalzo G. 32Carrara M. 60Carrupt P.-A. 38Chicca A. 53Choi H. 62

Index of Authors

Choi H.-Y. 81Cocucci M. 57Cola M. 64Cola-Miranda M. 76, 77Colombo M.L. 58, 59Conti A. 58, 59, 82, 84Corasaniti M.T. 36Cosentino M. 30, 55, 56,

57, 58, 59, 82Crema F. 55Dajas F. 39, 56Dall’Acqua S. 60de Paula Michelatto D. 77de Paula Vasconcelos P.C. 65Delle Canne M.G 55.Dietrich H. 23Distaso M. 74Domenici M.R. 32Echart C. 74Echeverri C. 39Farias-Silva E. 76, 77Ferrari M. 55Fink D. 25Fortinguerra S. 60Francescato P. 61Frank C. 32Frank N. 23Fratto V. 36Gagnier J.J. 46Gerhauser C. 23Gertsch J. 29Giorgi A. 57, 61Giovannini C. 34Girault J.-P. 72Gjorgoski I. 68Graziadio B. 74Greco R. 45Grogg A.F. 83Ham I. 62, 81

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Hiruma-Lima C.A. 63, 64, 65,76, 77

Ho R. 72Hostanska K. 21Hostettmann K. 17, 79Houghton P.J. 78Hylands P.J. 78Iacobelli M. 74Ibba F. 51Innocenti G. 60Iten F. 66Jun K. 62Jung E.-D. 81Kadifkova Panovska T. 68Karlina M. 75Kasture S. 67Kasture S.B. 51Klimo K. 23Ko Y. 62, 81Kulevanova S. 68Kushima H. 64Lafont R. 72Langer T. 18Lecchini S. 30, 55, 56,

57, 58, 59Lee B.-H. 81Lee J. 62, 81Legnaro M. 55Leu T. 73Licheri G. 57Lima Z.P. 63Longoni R. 67Loquet D. 72, 73Luini A. 30, 55, 56,

57, 58, 59Luiz-Ferreira A. 76, 77Luzzani M. 55Madeo M. 61Maida S. 36Makarov V. 69

86

Makarova M. 69Makarova M.N. 75Malchiodi-Albedi F. 32Malnoe P. 15Marcon N. 83Marino F. 30, 55, 56,

57, 58, 59, 82Marston A. 79Martel S. 38Martinotti E. 53Masella R. 34Massarelli I. 53Matteucci A. 32Meier R. 70Meijer L. 73Melzer J. 70Morazzoni P. 45Morelli M. 67Nappi G. 45Navarra M. 36Nieri P. 53Pakalapati G. 71Pan L. 23Paracchini S. 55Paradisi S. 32Park C. 62Park J. 62Pellati F. 53Pellizzon C.H. 63, 65Petrushevska G. 68Pinna A. 67Pontis S. 67Poretti Lozano Becerra G. 84Posner G.H. 25Pozharitskaya O. 75Raharivelomanana P. 72, 73Rasini E. 55Reddy M.N. 52Reichling J. 41Reist M. 38




Repice C. 74Rinaldo D. 64, 76Riva A. 45Rivera F. 39Rocha L.R.M. 64Rodrigues C.M. 64Rollinger J.M. 18Saller R. 66, 70Sandrini G. 45Santos L.C. 64Schlegel R. 25Schmeiser H.H. 42Schneele J. 41Schwambach Vieira A. 77Scorcia G. 32Selesneva A. 69Shikov A. 75Siddiqui B.S. 54Signorelli A. 60Silva E.F. 63Simola N. 67Simonnet X. 15Soulet S. 73Souza Brito A.R.M. 76, 77

Speranza G. 61Spiga S. 51Spina L. 67Stuppner H. 18Takayama C. 76Tassorelli C. 45Teai T. 72Techatanawat I. 78Tikhonov V.P. 75Urbain A. 79Usmani S.B. 54Vaamonde L. 39Vakharia B.C. 52Vilegas W. 63, 65, 76, 77Villegas W. 64Vinci S. 51Walt H. 44Wasilewski B.W. 80Will F. 23Wink M. 19, 71Yang G. 62, 81Zanni M. 82Zessner H. 23Zonnevijlle F. 83

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Under Auspices of

Comunità di Lavoro Regio Insubrica

Repubblica e Cantone Ticino

Provincia di Varese

Università degli Studi dell'Insubria

Ordine dei Medici Chirurghi e degli Odontoiatri della provincia di Varese

Ordine dei Medici e Odontoiatri di Como

Ordine dei Medici del Cantone Ticino

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